JP2004502134A - Receptor binding compounds and methods for their identification - Google Patents

Abstract

The present invention relates generally to G protein-coupled receptors ("GPCRs"), and more particularly to compounds that bind to G protein-coupled receptors. The methods of the invention are useful for treating diseases by identifying and preparing therapeutic compounds that bind to G protein-coupled receptors. The present invention provides compositions of binding compounds for G protein-coupled receptors and provides methods for identifying binding compounds for G protein-coupled receptors. The present invention also provides therapeutic agents containing such compounds.

Description

（実施例２：ＧＳＴタグ化ＣＣＲ５の調製およびスクリーニング）
ライブラリーを、Ｒｉｎｋ　Ａｍｉｄｅ　ＭＢＨＡ樹脂（置換：０．５４ｍｍｏｌ／ｇｍ）を使用して９−フルオレニルメトキシカルボニル（Ｆｍｏｃ）保護基と共に、ＡＢＩ　４３３Ａ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ，Ｆｏｒｓｔｅｒ　Ｃｉｔｙ，ＣＡ）上に合成した。縮重位置に対するアミノ酸のほぼ等しいカップリングを得るために、アミノ酸量を、文献値を考慮して経験的に調節した。例えば、Ｈｏｏｋ　Ｗ．ら、Ｆｅｄ．Ｐｒｏｃ．４４：１３２３（１９８５）を参照のこと。例示的なカップリング試薬は、１当量のペプチドに対して１当量の、ＨＢＴＵ／ＨＯＢＴ／ＤＩＥＡであった。開裂はカクテル（８２％　ＴＦＡ、５％フェノール、５％チオアニソール、２．５％　１，２−エタンジチオール、５％水）によって達成した。ペプチドをメチル−ｔｅｒｔ−ブチルエーテルから沈澱した。ライブラリーをＭＡＬＤＩ−ＴＯＦ　ＭＳ（Ｌｏｕｓｉａｎａ　Ｓｔａｔｅ　Ｕｎｉｖｅｒｓｉｔｙ）およびアミノ酸配列決定によって特徴づけた。
【００５０】
出発ライブラリーは、例えば、単一な非縮重指向化アミノ酸（すなわち、Ｘ−Ｘ−Ｘ−Ｘ−Ｒ−Ｘ−Ｘ−Ｘ−Ｘ）を使用した。２次ライブラリーは、いくつかの縮重位置に見出された最適な残基を固定化することにより調製し得る。例えば、Ｘ−Ｘ−Ｘ−Ｘ−Ｒ−Ｘ−Ｘ−Ｘ−Ｘは、第−４位がプロリンであるべきであり、その結果２次ライブラリーは、Ｐ−Ｘ−Ｘ−Ｘ−Ｒ−Ｘ−Ｘ−Ｘ−Ｘであることを示し得る。
【００５１】
レセプターのＧＳＴ形態を用いたペプチドライブラリーの使用を本明細書中で提供する。ＧＳＴタグ化ＧＰＣＲの場合、このレセプターはこのライブラリーに曝露され得、そして、遠心分離によりこれらの膜を、ペレットすることによって、遊離ペプチドと結合したペプチドとの分離を達成した。ＧＳＴタグ化精製レセプターをペプチドライブラリー、約１μｍｏｌｅのペプチドおよび約１ｎｍｏｌの結合部位と共にインキュベートした。インキュベーション後、結合したペプチドを伴ったレセプターを、遠心分離によって非結合ペプチドから分離した（ペレット中のレセプター・ペプチド複合体、上清中の非結合ペプチド）。非特異的に結合したペプチドを、徹底的な洗浄によって除去し得、そして低いｐＨ（２．５以下）のペレットの再懸濁物を使用して結合したペプチドを除去した。このペプチドを配列決定し、コンセンサス配列を決定した。
【００５２】
ＦＬＡＧ−タグ化ＧＰＣＲを、ペプチドライブラリースクリーニングのために使用する場合、このレセプターを抗ＦＬＡＧ　Ｍ２親和性マトリックス（Ｓｔ．Ｌｏｕｉｓ、ＭＯ）上に固定化した。さらなる精製アプローチはＧＳＴ構築物および固定したグルタチオン（Ｐｉｅｒｃｅ、Ｒｏｃｋｆｏｒｄ、ＩＬ）を使用し得た。
【００５３】
結合したペプチド混合物と出発ペプチドライブラリーの両方を、標準的な技術を使用して配列決定し得た。各アミノ酸量を、位置の関数として、決定した。各位置の各アミノ酸に対する優先値を、出発ライブライブラリーに存在するアミノ酸量とペプチドの結合画分とを比較することによって計算した。これらの手順を使用して、多くの結合ドメインと相互作用するペプチドの好ましい配列を生成し、そしてこれを表１に記載した。
【００５４】
また、２次ライブラリーを出発ライブラリーからの情報を組み込むことによって配列決定し得た。特徴付けのこの第１のラウンドについて、ファージディスプレイ技術をまた使用して予備的な結合モチーフを同定する。ファージディスプレイ法は、天然のアミノ酸のモチーフの同定を提供する。ファージディスプレイ技術は、ＤＮＡ配列を、ファージ被覆タンパク質の１つをコードする遺伝子中へ挿入することを包含する。この遺伝子を特定の位置に挿入して、その結果発現されたタンパク質挿入物は他の分子と相互作用し得る。結果として、コードされたペプチドまたはタンパク質配列を、ファージ表面上に提示し、そして結合のために曝露する。縮重ヌクレオチドを挿入することによって、各ファージは、異なったペプチド配列を発現し得る（「ファージライブラリー」）。このファージライブラリーと固定したレセプターとのインキュベーションを使用して、このレセプターに特異的に結合する配列を同定し得る。シグナルは、単離されたファージを増殖することよって増幅することができるので、微弱なシグナルでさえ検出し得る。ファージディスプレイから導出した情報は、薬学的特徴を増大したリガンドを選択するために、新規のアミノ酸アナログまたは環状ペプチドを含む合成ライブラリーを使用する、親和性精製法に対して適用可能である。出発ライブラリー、２次ライブラリーおよび３次ライブラリーの使用によって、結合部位の特異性の完全な定義を提供した。
【００５５】
一旦、好ましいアミノ酸残基を、このライブラリーの縮重位置における、高い優先値を使用して同定すると、特異的なペプチドをライブラリー合成に用いたような方法によって合成し得た。これらをＨＰＬＣによって精製し、そして組成をＭＡＬＤＩ−ＴＯＦ　ＭＳによって確認した。
【００５６】
相対的な親和性を、レセプター精製に使用したヨウ素化結合アッセイを改変することによって測定し得る。従って、この精製したレセプター膜から［^１２５Ｉ］−リガンドを提示するこれらのペプチドの能力を測定し得た。しかし、この天然リガンドが既知でない場合、この活性を当該分野で標準的な細胞に基いたアッセイを使用して測定し得る。また、会合定数を当該分野で標準的な技術を使用して決定し得る。
【００５７】
（実施例３：タグ化したＣＣＲ５の調製と分析およびこれのライブラリースクリーニング）
（Ａ．ＣＣＲ５のクローニングおよび発現）
ＣＣＲ５　ｃＤＮＡ（図４の配列を参照）をベクターｐＣＤＮＡ３中のＲｅｃｅｐｔｏｒ　Ｂｉｏｌｏｇｙ（Ｂｅｌｔｓｖｉｌｌｅ，ＭＤ）から得た。タグを、レセプターのＣ末端に加え、本明細書中に記載のような、標準的な技術を使用する親和性精製アッセイのために、レセプターを固定するのに使用した。さらに、ＧｅｎＢａｎｋ　登録番号Ｕ５７８４０に相関するＣＣＲ５　ｃＤＮＡを、ベクターｐＣＤＮＡ３中で使用し得る。Ｒｅｃｅｐｔｏｒ　Ｂｉｏｌｏｇｙ由来のＣＣＲ５　ｃＤＮＡと、ＧｅｎＢａｎｋ　登録番号Ｕ５７８４０に相関するＣＣＲ５　ｃＤＮＡとの間の唯一の違いは、第１８０位の塩基の点変異（このアミノ酸配列において、ロイシンをグルタミンに変化させる）である。以下は、このタグ化方法を使用した実験からの具体的な例である：
（Ｃ末端ヒスチジンタグを伴ったＣＣＲ５の構築（昆虫選択発現系））
ＣＣＲ５−ＨＩＳ構築物をこの系から誘導した。ＰＣＲをＣＣＲ５／ｐｃＤＮＡ５（Ｒｅｃｅｐｔｏｒ　Ｂｉｏｌｏｇｙ製）をテンプレートとして使用して、そしてプライマー５−Ａｇｅ　Ｈｉｓおよびプライマー５−Ｓｐｅ　Ｈｉｓを使用して実施した。この最初のプライマーによって、独特のＳｐｅＩ部位を、ＣＣＲ５の開始ＡＴＧの直前に誘導した。第２のプライマーによって、ＣＣＲ５の終止コドンを、ｐＩＺＴ／Ｖ５−Ｈｉｓ（Ｉｎｖｉｔｒｏｇｅｎ、Ｃａｒｌｓｂａｄ、ＣＡ）のヒスチジンタグとインフレームであるＡｇｅＩ部位の中へ変異した。ＰＣＲ産物をＳｐｅＩとＡｇｅＩで消化し、次いで、同様に消化されたｐＩＺＴ／Ｖ５−Ｈｉｓの中へ連結した。この構築物は、ＣＣＲ５−Ｈｉｓ−ＰＩＺＴとして同定される。
【００５８】
（Ｃ末端ヒスチジンタグを伴ったＣＣＲ５の構築（バキュロウイルス発現系））
ＣＣＲ５−Ｈｉｓ−ＰＩＺＴをＨａｅＩＩとＳｐｅＩで消化し、次いで、クレノウフラグメントで埋めた。ＣＣＲ５−Ｈｉｓを含むこのフラグメントを、予めＮｈｅＩで消化され、そしてクレノウで平滑化したｐＢｌｕｅｂａｃ　４．５（Ｉｎｖｉｔｒｏｇｅｎ）中へ、連結した。この構築物を方向の正確さについてチェックした。これは、ＣＣＲ５−ＨＩＳと呼ばれる。この構築物を、制限消化および標準的な技術を用いた配列決定によって確認した。この構築物を発現のために使用し、そして十分に発現され、そして親和性精製スクリーニングでの使用のための活性形態にあることを決定した。
【００５９】
（Ｃ末端ＦＬＡＧタグを有するＣＣＲ５の構築）
標準的な技術を使用するＰＣＲを、テンプレートとしてＣＣＲ５／ｐｃＤＮＡ３（Ｒｅｃｅｐｔｏｒ　Ｂｉｏｌｏｇｙ製）ならびにプライマー５−Ｘｈｏ　ｐＦＬＡＧおよびプライマー５−Ｓａｌ　ｐＦＬＡＧを使用して実施した。第１のプライマーは、ＣＣＲ５のイニシエーターＡＴＧの直前の固有のＸｈｏＩ部位を操作した。第２のプライマーは、ＣＣＲ５の停止コドンをＳａｌＩ制限部位に変異した（Ｓｉｇｍａ製のｐＦＬＡＧ−ＣＴＣのＦＬＡＧタグを用いてインフレームで）。ＰＣＲ産物を、ＸｈｏＩおよびＳａｌＩで消化し、そして同様に消化したｐＦＬＡＧ−ＣＴＣ（細菌の発現ベクター）に連結した。この構築物を、ＣＣＲ５−ＦＬＡＧ−ＣＴＣと呼ぶ。次いで、ＣＣＲ５−ＦＬＡＧを、ＸｈｏＩおよびＳｃａＩで消化し、そしてクレノウ断片で充填した。ＣＣＲ５−ＦＬＡＧ−ＣＴＣを含むフラグメントを、最初にＮｈｅＩで消化し、次いで、クレノウで平滑化したｐＢｌｕｅｂａｃ４．５に連結した。この最終構築物を、ＣＣＲ５−ＦＬＡＧとして同定する。この構築物を、制限消化によって確認し、そして標準的な技術を使用して配列決定した。この構築物を、発現のために使用し、そしてアフィニティー精製スクリーニングにおける使用のために十分にかつ活性な形態で発現することを決定した。
【００６０】
（Ｃ末端ＧＳＴタグを有するＣＣＲ５の構築）
ＰＣＲを、テンプレートとしてＣＣＲ５／ｐｃＤＮＡ３（Ｒｅｃｅｐｔｏｒ　Ｂｉｏｌｏｇｙ製）ならびにプライマーＧＳＴ−ＢａｍＨＩおよびプライマーＧＳＴ−ＮｄｅＩを使用して実施した。第１のプライマーを、ＣＣＲ５の停止コドンを、ＢａｍＨＩ制限部位に変異した（ｐＥＳＰ−３のＦＬＡＧ／ＧＳＴタグを用いてインフレームで）。第２のプライマーを、ＣＣＲ５の開始コドンにおける固有のＮｄｅＩ部位に導入した。ＰＣＲ産物を、ＢａｍＨＩおよびＮｄｅＩで消化し、そして同様に消化したｐＥＳＰ−３（酵母発現ベクター）に連結した。この構築物を、ｐＣＰ８と呼ぶ。次いで、ｐＣＰ８を、ＮｄｅＩおよびＳｍａＩで消化し、そしてクレノウ断片で充填した。ＣＣＲ５−ＧＳＴを含むフラグメントを、最初にＢｇｌＩＩで消化し、次いでクレノウで平滑化した、ｐＢｌｕｅｂａｃ４．５に連結した。この最終構築物を、ｐＣＰ１０として同定する。このタンパク質を記載する場合、この構築物を、ＣＣＲ５−ＧＳＴとして同定する。この構築物を、制限消化によって確認し、そして標準的な技術によって配列決定した。この構築物を、発現のために使用し、そしてアフィニティー精製スクリーニングにおける使用のために十分にかつ活性な形態で発現することを判定した。
【００６１】
３つの新しい構築物のためのベクター（ＣＣＲ５−ＦＬＡＧ、ＣＣＲ５−ＧＳＴおよびＣＣＲ５−ＨＩＳ）を使用して、それぞれのウイルスストックの産生のためにＳｆ９細胞を同時トランスフェクトした。これらのウイルスストックを、標準的なプラークアッセイを使用して精製し、次いで実験に使用して、種々のＣ末端タグを有するＣＣＲ５の発現の最適化のために感染させた。Ｈｉｇｈ　Ｆｉｖｅ細胞（Ｉｎｖｉｔｒｏｇｅｎ）をまた、これらのＣＣＲ５タグ化構築物でトランスフェクトし、そしてＣＣＲ５の発現について試験した。全ての構築物について、適切なタグ化レセプターを発現することを判定した。７２時間後の発現レベルは、Ｓｆ９細胞について発現レベルよりもＨｉｇｈ　Ｆｉｖｅ細胞において５倍ほど高かった。全ての上記の実験を、当業者に公知の標準的な技術を使用して行なった。
【００６２】
ＣＣＲ５の発現のための第４の構築物を、スターティングベクターｐＢｌｕｅＢａｃ４．５（Ｉｎｖｉｔｒｏｇｅｎ）から作製し、以前記載したベクターにおけるトロンビン開裂部位およびエンテロキナーゼ開裂部位を除去した。ＧＳＴタグを、ＰＣＲを使用することによってマルチクローニング部位に付加してＧＳＴタグを精製し、次いで、当業者に公知の標準的な方法を使用して消化したベクター（ＳｍａＩ／ＥｃｏＲＩ）に連結した。次に、このベクターを、操作の容易化のためにＬｉｆｅｔｅｃｈ製のＧａｔｅｗａｙ技術と適合性にした。これを、この技術のために必要な組換え部位を含むカセット（Ｌｉｆｅｔｅｃｈ製）にＳｍａＩを連結することによって行なった。ＣＣＲ５を、プライマーを使用してＰＣＲを用いて増幅して、組換えのための結合部位を含む遺伝子を伸長した。次いで、ＰＣＲ産物を、ＢＰクロナーゼ（相同組換えに必要な酵素）を使用してバキュロウイルスベクターに組み込んで、エンテロキナーゼ開裂部位またはトロンビン開裂部位を有さず、Ｃ末端ＧＳＴタグ有するＣＣＲ５を含むバキュロウイルス発現のためのベクターを作製した。このベクターを、発現のために必要なウイルスストックの調製のためにＳｆ９細胞に同時トランスフェクトした。このウイルスをプラーク精製し、そしてＰＣＲおよび配列チェックしたクローンを、ＣＣＲ５の発現のために使用した。この構築物のタイムコースは、タンパク質のより少ないのタンパク質分解が観察され、そしてより少ない時間が、レセプターの最大の発現を得るために必要であったことを示した。
【００６３】
（Ｂ．ＣＣＲ５の活性）
それぞれのタグ化ＣＣＲ５遺伝子（ＣＣＲ５−ＧＳＴ、ＣＣＲ５−ＦＬＡＧおよびＣＣＲ５−ＨＩＳ）を、本明細書中に記載されるように、Ｓｆ９細胞およびＨｉｇｈ　Ｆｉｖｅ細胞において発現させた。全てのＳｆ９細胞株由来の全細胞およびＨｉｇｈ　Ｆｉｖｅ細胞株由来の全細胞を、低張緩衝液（１０ｍＭ　Ｔｒｉｓ（ｐＨ７．４）、５ｍＭ　ＥＤＴＡ）を使用して溶解し、そして膜調製物を、当業者に公知の標準的な技術を使用するホモジナイズおよび遠心分離によって作製した。ＣＣＲ５−ＧＳＴ、ＣＣＲ５−ＦＬＡＧおよびＣＣＲ５−ＨＩＳに関する膜調製物を、それぞれ、標準的な放射リガンド結合アッセイを使用してアッセイした。結合アッセイを、５０ｍＭのＨｅｐｅｓ（ｐＨ７．５）、１％　ＢＳＡ中の５μｇの膜を用いて実施した。放射リガンドＭＩＰ１−β（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｎｕｃｌｅａｒ、「ＮＥＮ」から得た）を、冷ＭＩＰ１α（競合リガンド；ＣＣＲ５に対する天然のリガンドは、ＲＡＮＴＥＳ、ＭＩＰ１βおよびＭＩＰ１αである）と共に、および冷ＭＩＰ１αなしで室温で１時間、膜と共にインキュベートし、濾過し、洗浄し、そして結合した放射活性計数を、シンチレーションカウンターを使用して測定した。非トランスフェクト細胞を、この実験のコントロールとして使用した。膜調製物の活性は、少なくとも２０％の活性タンパク質を有するＲｅｃｅｐｔｏｒ　Ｂｉｏｌｏｇｙ（ＭＩＰ１−β結合に関してＫ_ｄ＜１ｎＭ）によって得られた膜調製物に匹敵した。
【００６４】
（Ｃ．ＣＣＲ５の可溶化）
溶解した全細胞調製物および膜調製物の両方を、可溶化のために使用した。ＣＣＲ５（ＣＣＲ５−ＦＬＡＧ、ＣＣＲ５−ＧＳＴおよびＣＣＲ５−ＨＩＳ）のタグ化バージョンの可溶化を、約６．８〜約８．２の範囲のｐＨで、界面活性剤（例えば、ＮＰ−４０、ＴｒｉｔｏｎＸ−１００、β−Ｄ−マルトシド、ｎ−オクチルグルコシド、ＣＹＭＡＬ、Ｚｗｉｔｔｅｒｇｅｎｔｓ、Ｔｗｅｅｎ−２０、ライソホスファチジルコリン、ＣＨＡＰＳなど）、塩（例えば、ＮａＣｌ、ＣａＣｌ_２、ＭｇＣｌ_２、ＭｎＣｌ_２、ＫＣｌなど）、緩衝液（例えば、Ｔｒｉｓ、Ｈｅｐｅｓ、Ｈｅｐｐｓ、Ｐｉｐｅｓ、Ｍｅｓ、Ｍｏｐｓ、酢酸、リン酸、イミダゾールなど）の多くの異なる組み合わせを使用して実施した。最適な可溶化のための条件を、ｐＨ８．１でＺｗｉｔｔｅｒｇｅｎｔ３〜１４および低塩（例えば、低いマグネシウムおよびカルシウムであって、ＮａＣｌはない（０．０ｎＭ　ＮａＣｌ）を使用して見出した。好ましい実施形態において、少なくとも２０％の可溶化された、固定化タンパク質は、活性である。非常に好ましい実施形態において、少なくとも３０％、４０％、５０％および７５％の可溶化された固定化タンパク質は、活性である。
【００６５】
（Ｄ．ＣＣＲ５の固定化）
可溶化後、ＣＣＲ５−ＧＳＴとＣＣＲ５−ＦＬＡＧの両方を、精製のためおよびペプチドライブラリーのスクリーニングのための活性タンパク質として使用するためにアフィニティーカラムに固定化した。ライブラリーからのアフィニティー精製のためのＧＰＣＲの固定化を示す概略図を図３に示す。ＣＣＲ５−ＧＳＴを、グルタチオン−アガロース（Ｐｉｅｒｃｅ）およびグルタチオン−セファロース（Ａｍｅｒｓｈａｍ　Ｐｈａｒａｍａｃｉａ　Ｂｉｏｔｅｃｈ）に結合し、そして固定化し、そしてＣＣＲ５−ＦＬＡＧを、ＦＬＡＧエピトープを認識する特異的な抗体カラム（Ｍ２カラム，Ｓｉｇｍａ）に固定化した。活性形態におけるタンパク質の固定化は、カラムにある間、活性を維持するための界面活性剤ならびに適切なｐＨおよび適切な塩状態を必要とした。この活性を、放射標識化ＭＩＰ−１β（上記の膜アッセイに用いた）および冷ＭＩＰ−１αを用いた競合を使用して放射活性結合によって測定した。トランスフェクトされていない細胞を、カラム単独と同様に、この活性のためのコントロールとして使用した。これらの実験は、活性形態の樹脂に（アフィニティー精製スクリーニングに十分な）精製形態のマイクログラム量のレセプターを固定する能力を実証した。
【００６６】
（Ｅ．ペプチドライブラリー合成）
ライブラリーを、Ｒｉｎｋ　Ａｍｉｄｅ　ＭＢＨＡ樹脂（置換：０．５４ｍｍｏｌ／ｇｍ）を使用して９−フルオレニルメトキシカルボニル（Ｆｍｏｃ）保護基を有するＡＢＩ　４３３Ａ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ，Ｆｏｒｓｔｅｒ　Ｃｉｔｙ，ＣＡ）において合成した。アミノ酸の混合物を、位置を縮重するために使用する場合、およそ等量のアミノ酸の結合を、文献値を考慮にいれた後、経験的にアミノ酸の量を調節することによって得た（例えば、Ｉｖａｎｅｔｉｃｈら，Ｃｏｍｂｉｎａｔｏｒｉａｌ　Ｃｈｅｍｉｓｔｒｙ，第２６７巻，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ，Ｓａｎ　Ｄｉｅｇｏ，ＣＡ　ＵＳＡ，２４７〜２６０頁（１９９６）を参照のこと）。結合剤は、ＨＢＴＵ／ＨＯＢＴ／ＤＩＥＡ（等価のペプチドあたり１当量）であった。切断を、カクテル（８２％　ＴＦＡ、５％　フェノール、５％　チオアニソール、２．５％　１，２−エタンジチオール、５％　水）によって行なった。ペプチドは、メチル三級ブチルエーテルから沈殿させた。ライブラリーを、ＭＡＬＤＩ−ＴＯＦ　ＭＳ（Ｌｏｕｉｓｉａｎａ　Ｓｔａｔｅ　Ｕｎｉｖｅｒｓｉｔｙ）およびアミノ酸配列決定によって特徴付けた。
【００６７】
第１のライブラリーは、単一、縮重塩基性ベースアミノ酸（すなわち、Ｍ−Ｘ−Ｘ−Ｘ−Ｘ−Ｗ−Ｘ−Ｘ−Ｘ−Ｘ−Ａ−Ｋ−Ｋ−Ｋ）を使用した。これらの第１のライブラリーの使用を介して、幾つかのまたは全ての縮重位置における最適な残基を、規定した。第２のライブラリーを、全ての位置を規定していなかった場合、作製し、それによって規定の位置を固定した。
【００６８】
（Ｆ．固定化ＣＣＲ５を使用するペプチドライブラリーのスクリーニング）
特定の樹脂に固定化されたタンパク質を用いて（例えば、ＣＣＲ５−ＧＳＴをグルタチオン−セファロースにかまたはＣＣＲ５−ＦＬＡＧをＭ２抗体−アガロースに固定化する）、無数の化合物のスクリーニングを、一緒にインキュベートすること、およびＣＣＲ５によって好まれるリガンドを精製する自然の選択および結合親和性を可能にすることによって行い得る。これらの実験を、直鎖状ライブラリー４Ｐ４（＋）および他のライブラリーを使用して実施した。
【００６９】
（Ｇ．ファージディスプレイ）
スクリーニングにおいて有用な代替の方法またはさらなる方法として、固定された機能的ＣＣＲ５を使用して、当業者に公知の標準的な技術を使用してＣＣＲ５に結合するファージを単離した。アッセイにおいて使用されたグルタチオン−セファロースビーズ、ＢＳＡおよびＭＩＰ１βからのバックグラウンドのサブトラクションを、これらの化合物の混合物と共にファージライブラリーをインキュベートすることによって実施した。レセプターとの、サブトラクションしたファージライブラリー（すなわち、ＮＥＢ、ＰｈＤ　Ｃ７Ｃ）のインキュンベーション後、結合したファージを、天然のＣＣＲ５リガンド（ＭＩＰ−１β）およびグリシン、ｐＨ２．２の両方とによって溶出した。ＰｈＤ　Ｃ７Ｃは、ジスルフィド間に７個の無作為なアミノ酸を有する特殊なファージライブラリーであり、そしてＮｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ（「ＮＥＢ」）から得られ得る。複数回のスクリーニングを実施した。両方の条件は、ＣＣＲ５に結合し、そしてリガンド結合を阻害し得る特定の配列を提供した。
【００７０】
（実施例４：タグ化ＣＸＣＲ４の調製および分析ならびにそのライブラリースクリーニング）
（Ａ．ＣＸＣＲ４のクローニングおよび発現）
ＣＸＣＲ４を、二等分した脾臓ｃＤＮＡライブラリーから単離し、そして一緒にスプライスした。これらの２つのフラグメントを、ＰＣＲ技術ならびに３’末端および５’末端に対するプライマーならびにＣＸＣＲ４遺伝子の中央部を使用して単離した。全長クローンを、３’プライマーおよび５’プライマーを使用して単離しなかったが；しかし、２等分を、単離し、そして遺伝子内の固有のＢａｍＨＩ部位を使用して一緒に連結した。構築物の同定を、配列決定によって確認した。選択的スプライス短縮形態もまた、単離した。これを、ＣＸＣＲ４短縮型と呼ぶ。タグを、レセプターのＣ末端に付加し、標準的な技術を使用するアフィニティー精製アッセイのためにそれらを固定するために使用した。以下は、このタグ化方法を使用した実験からの具体的な実施例である。
【００７１】
（Ｃ−末端ヒスチジンタグを有するＣＸＣＲ４の構築（昆虫選択発現系））
ＧｎＲＨＲ（性腺刺激ホルモン放出ホルモンレセプター）に関する遺伝子を含む以前の構築物を、使用して、第１のＣＸＣＲ４構築物を作製した。ＧｎＲＨＲに関する遺伝子を、スプライスし、そしてＣＸＣＲ４に関する単離したｃＤＮＡによって置換した。このベクターは、本来、Ｃ末端に６×Ｈｉｓタグを有するｐｅｔ３０ａベクターであった。
【００７２】
（Ｃ末端ＦＬＡＧタグを有するＣＸＣＲ４構築物の構築）
ＰＣＲを、プライマー５’ＢｓｐＥ１　ＣＸＣＲ４およびプライマー３’Ｂｇｌ　ＣＸＣＲ４を使用して実施し、そしてＳｉｇｍａ製のｐＦＬＡＧ−ＣＴＣ（細菌発現ベクター）のＦＬＡＧタグを有するインフレームＣＸＣＲ４の連結のための固有の部位を操作した。この構築物を、ＣＸＣＲ４−ＦＬＡＧ−ＣＴＣと呼ぶ。次いで、ＣＸＣＲ４−ＦＬＡＧを、消化によって除去し、そしてクレノウ断片で充填した。ＣＸＣＲ４−ＦＬＡＧを含むフラグメントを、最初に、消化し、次いで、クレノウで平滑化したｐＢｌｕｅｂａｃ４．５に連結した。この最終構築物を、ＣＸＣＲ４−ＦＬＡＧと呼ぶ。この構築物を、制限消化によって確認し、そして標準的な技術を使用して配列決定した。この構築物を、発現のために使用し、そしてアフィニティー精製スクリーニングにおける使用のために十分にかつ活性な形態で発現することを判定した。
【００７３】
（Ｃ末端ＧＳＴタグを有するＣＸＣＲ４構築物の構築）
新しく構築されたＣＸＣＲ４−ＦＬＡＧ　ｃＤＮＡを、ＣＴＣベクターから除去し、そしてＣＣＲ５の代わりに（ＢｇｌおよびＢｓｐＥ１を使用して）別の構築物（ＣＣＲ５−ＧＳＴ）にサブクローニングした。これは、１工程を使用してＣＸＣＲ４−ＧＳＴのためのベクターを作製した。の構築物を、制限消化によって確認し、そして標準的な技術を使用して配列決定した。この構築物を、発現のために使用し、そしてアフィニティー精製スクリーニングにおける使用のために十分にかつ活性な形態で発現することを判定した。
【００７４】
（Ｎ末端６×Ｈｉｓタグを有するＣＸＣＲ４の構築）
この構築物を市販のベクター、ｐＢｌｕｅｂａｃＨｉｓ２ｂ（Ｉｎｖｉｔｒｏｇｅｎ）にＣＸＣＲ４をサブクローニングすることにより調製した。この構築物を、標準的技術を用いた制限消化および配列決定により確認した。
【００７５】
３つの新規の構築物、ＣＸＣＲ４−ＦＬＡＧ、ＣＸＣＲ４−ＧＳＴ、およびＣＸＣＲ４−ＨＩＳのベクターを用いて、それぞれのウイルスストックの作成のためにＳｆ９細胞を同時トランスフェクトした。これらのウイルスストックを、標準的なプラークアッセイを用いて精製し、次いでその種々のＣ末端タグを有するＣＸＣＲ４の発現の最適化に関する感染実験において用いた。Ｈｉｇｈ　Ｆｉｖｅ細胞（Ｉｎｖｉｔｒｏｇｅｎ）もまた、これらのＣＸＣＲ４タグ化構築物を用いてトランスフェクトし、そしてＣＸＣＲ４の発現について試験した。全ての構築物を適切にタグ化したレセプターを発現するために決定した。Ｈｉｇｈ　Ｆｉｖｅ細胞における７２時間後の発現レベルは、Ｓｆ９細胞におけるレベルよりも５倍高かった。
【００７６】
（Ｂ．ＣＸＣＲ４の活性）
それぞれのタグ化ＣＸＣＲ４遺伝子（ＣＸＣＲ４−ＦＬＡＧ、ＣＸＣＲ４−ＧＳＴ、およびＣＸＣＲ４−ＨＩＳ）を、本明細書中に記載される、Ｓｆ９およびＨｉｇｈ　Ｆｉｖｅ細胞に同時トランスフェクションするために用いた。Ｓｆ９およびＨｉｇｈ　Ｆｉｖｅ細胞株由来の全細胞を、低張緩衝液（１０ｍＭ　Ｔｒｉｓ（ｐＨ７．４）、５ｍＭ　ＥＤＴＡ）を使用して溶解し、そして膜調製物を、当業者に公知の標準的な技術を使用するホモジナイズおよび遠心分離によって作製した。ＣＸＣＲ４−ＧＳＴ、ＣＸＣＲ４−ＦＬＡＧおよびＣＸＣＲ４−ＨＩＳについての膜調製物を、標準的な放射性リガンド結合アッセイを使用してアッセイした。放射リガンド［^１２５Ｉ］−ＳＤＲ−１（ＮＥＮ）を、冷ＳＤＦ−１（競合する天然のリガンド）と共に、およびそれを伴わずに、室温にて１時間、膜と共にインキュベートし、濾過し、洗浄し、そして結合した放射性計数をシンチレーション計数を用いて検出した。非トランスフェクト細胞を、この実験のコントロールとして使用した。膜調製物の活性は、少なくとも２０％の活性タンパク質を生じた。
【００７７】
（Ｃ．ＣＸＣＲ４の可溶化）
溶解した全細胞および膜調製の両方を、可溶化のために使用した。ＣＸＣＲ４にタグ化バージョン（ＣＸＣＲ４−ＦＬＡＧ、ＣＸＣＲ４−ＧＳＴおよびＣＸＣＲ４−ＨＩＳ）の可溶化を、界面活性剤（例えば、ＮＰ−４０、ＴｒｉｔｏｎＸ−１００、β−Ｄ−マルトシド、ｎ−オクチルグルコシド、ＣＹＭＡＬ、Ｚｗｉｔｔｅｒｇｅｎｔｓ、Ｔｗｅｅｎ−２０、ライソホスファチジルコリン、ＣＨＡＰＳなど）、塩（例えば、ＮａＣｌ、ＣａＣｌ_２、ＭｇＣｌ_２、ＭｎＣｌ_２、ＫＣｌなど）、緩衝液（例えば、Ｔｒｉｓ、Ｈｅｐｅｓ、Ｈｅｐｐｓ、Ｐｉｐｅｓ、Ｍｅｓ、Ｍｏｐｓ、酢酸、リン酸、イミダゾールなど）の多くの異なる組み合わせを約６．８〜約８．２の範囲のｐＨにて使用して実施した。最適な可溶化のための条件を、ｐＨ８．１でＺｗｉｔｔｅｒｇｅｎｔ３〜１４および低塩（例えば、低いマグネシウムおよびカルシウムであって、ＮａＣｌはない（０．０ｎＭ　ＮａＣｌ）を使用して見出した。好ましい実施形態において、少なくとも２０％の可溶化された、固定化タンパク質は、活性である。非常に好ましい実施形態において、少なくとも３０％、４０％、５０％および７５％の可溶化された、固定化タンパク質は、活性である。
【００７８】
（Ｄ．ＣＸＣＲ４の固定化）
可溶化後、ＣＸＣＲ４−ＧＳＴを、精製のためおよびペプチドライブラリーのスクリーニングにおける使用のための活性タンパク質として使用するためにアフィニティーカラムに固定化した。ライブラリーからのアフィニティー精製のためのＧＰＣＲの固定化の示す概略図を、図３に示す。ＣＸＣＲ４−ＧＳＴを、グルタチオン−アガロース（Ｐｉｅｒｃｅ）およびグルタチオン−セファロース（Ａｍｅｒｓｈａｍ　Ｐｈａｒａｍａｃｉａ　Ｂｉｏｔｅｃｈ）に結合し、そして固定化した。活性形態のタンパク質の固定化は、カラム上に置かれる間、活性形態を維持するために界面活性剤の使用ならびに適切なｐＨおよび塩濃度を必要とした。この活性を、放射標識したＳＤＦ−１（上記の膜アッセイを伴う場合）放射性結合および冷ＳＤＦ−１との競合により決定した。トランスフェクトされていない細胞を、カラム単独と同様に、この活性のためのコントロールとして使用した。これらの実験は、活性形態の樹脂に、アフィニティー精製スクリーニングに十分な精製形態のマイクログラム量のレセプターを固定する能力を実証した。
【００７９】
（Ｅ．ペプチドライブラリー合成）
ライブラリーを、Ｒｉｎｋ　Ａｍｉｄｅ　ＭＢＨＡ樹脂（置換：０．５４ｍｍｏｌ／ｇｍ）を使用して９−フルオレニルメトキシカルボニル（Ｆｍｏｃ）保護基を有するＡＢＩ　４３３Ａ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ，Ｆｏｒｓｔｅｒ　Ｃｉｔｙ，ＣＡ）において合成した。アミノ酸の混合物を、位置を縮重位置について使用する場合、およそ等量のアミノ酸の結合を、文献の値を考慮にいれた後、経験的にアミノ酸の量を調節することによって得た（例えば、Ｉｖａｎｅｔｉｃｈら，Ｃｏｍｂｉｎａｔｏｒｉａｌ　Ｃｈｅｍｉｓｔｒｙ，第２６７巻，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ，Ｓａｎ　Ｄｉｅｇｏ，ＣＡ　ＵＳＡ，２４７〜２６０頁（１９９６）を参照のこと）。結合剤は、ＨＢＴＵ／ＨＯＢＴ／ＤＩＥＡ（等価のペプチドあたり１当量）であった。切断を、カクテル（８２％　ＴＦＡ、５％　フェノール、５％　チオアニソール、２．５％　１，２−エタンジチオール、５％　水）によって行なった。ペプチドは、メチル三級ブチルエーテルから沈殿した。ライブラリーを、ＭＡＬＤＩ−ＴＯＦ　ＭＳ（Ｌｏｕｉｓｉａｎａ　Ｓｔａｔｅ　Ｕｎｉｖｅｒｓｉｔｙ）およびアミノ酸配列決定によって特徴付けた。
【００８０】
第１のライブラリーは、単一、非縮重塩基性ベースアミノ酸（すなわち、Ｍ−Ｘ−Ｘ−Ｘ−Ｘ−Ｗ−Ｘ−Ｘ−Ｘ−Ｘ−Ａ−Ｋ−Ｋ−Ｋ）を使用した。これらの第１のライブラリーの使用を介して、幾つかのまたは全ての縮重位置における最適な残基を、規定した。第２のライブラリーを、全ての位置を規定していなかった場合、作製し、それによって規定の位置を固定した。
【００８１】
（Ｆ．固定化ＣＸＣＲ４を使用するペプチドライブラリーのスクリーニング）　タンパク質を、特定の樹脂に固定化し（例えば、ＣＸＣＲ４−ＧＳＴをグルタチオン−セファロースに固定化する）、無数の化合物のスクリーニングを、一緒にインキュベートすること、およびＣＸＣＲ４に好まれるリガンドを精製する自然の選択および結合親和性を可能にすることによって行い得る。これらの実験を、１０のライブラリーを使用して実施した。そしてこれらの実験は、他のライブラリーを用いて実施し得る。
【００８２】
（Ｇ．ファージディスプレイ）
スクリーニングにおいて有用な代替の方法またはさらなる方法として、固定された機能的ＣＸＣＲ４を使用して、当業者に公知の標準的な技術を使用してＣＸＣＲ４に結合するファージを単離した。アッセイにおいて使用されたグルタチオン−セファロースビーズ、ＢＳＡおよびＳＤＦ１αからのバックグラウンドのサブトラクションを、これらの化合物の混合物と共にファージライブラリーをインキュベートすることによって実施した。レセプターとの、サブトラクションしたファージライブラリー（すなわち、ＮＥＢ　ＰｈＤ　Ｃ７Ｃ）のインキュンベーション後、結合したファージを、天然のＣＸＣＲ４リガンド（ＳＤＦ１α）およびグリシン、ｐＨ２．２の両方とによって溶出した。ＰｈＤ　Ｃ７Ｃは、ジスルフィド間に７個の無作為なアミノ酸を有する特殊なファージライブラリーであり、そしてＮｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ（「ＮＥＢ」）から得られ得る。複数回のスクリーニングを実施した。両方の条件は、ＣＸＣＲ４に結合し、そしてリガンド結合を阻害し得る特定の配列を提供した。
【００８３】
（実施例５：疾患および障害の予防または処置）
しかし、現在、治療用ペプチド、ペプチド模倣物、または疾患および障害（例えば、ＡＩＤＳおよびＨＩＶ感染）の予防および処置のために使用されるＧＰＣＲ結合の低分子アゴニストもしくはアンタゴニストの生産、処方および使用の間に、いくつかの複雑な問題が生じる。ＧＰＣＲの治療的結合化合物の商業的生産のコストおよび技術的難易度を最小化する生物学的に適切なアンタゴニストまたはアゴニストはさらに、本発明によって意図される。さらに、アンタゴニストまたはアゴニストに対する免疫学的応答を与えず、その結果その有効性を妨害する、ＧＰＣＲ結合の生物学的に適切なアンタゴニストまたはアゴニストは、本発明によって意図される。さらに、生成されたＧＰＣＲ結合のアンタゴニストまたはアゴニストの、生物学的効力の有意な喪失を伴わない、商業的に妥当な有効期限を与える適切な処方は、本発明において意図される。さらに、ＡＩＤＳおよびＨＩＶ感染のような疾患および状態の予防および処置のために適切な、個体に対する投与のための有用な用量は、本発明において意図される。
【００８４】
単独でまたは他の適切な分子と混合した、ＧＰＣＲ結合の阻害に関してコンピテントな適切な候補物の同定は、本発明によって意図される。このような候補物はさらに、ＡＩＤＳおよびＨＩＶ感染のような疾患および状態の予防および処置のために生物学的に適切な、上記で同定された候補物の商業的に重要な量の生産を意図する。さらに、本発明は、最初に同定されたアナログの所望されない任意の特性（例えば、インビボでの毒性または貯蔵の際の分解傾向）が緩和される、ＧＰＣＲ結合アンタゴニスト、アゴニストまたは誘導体の治療的品質の商業的に重要な量の生産を提供する。
【００８５】
ペプチド、ペプチド模倣物、またはＧＰＣＲ結合活性の低分子アンタゴニストの同定および設計後の、ＡＩＤＳおよびＨＩＶ感染のような疾患を予防および処置する方法は、本明細書中に記載のようなＧＰＣＲ結合を阻害し得る化合物を含む組成物を投与する工程を包含する。投与は、任意の適合性の経路によるものであり得る。従って、適切な場合、投与は、経口または非経口（投与の静脈内および腹腔内の投与経路を含む）を含み得る。特に好ましい方法は、適切な処方物の徐放性注射によるものである。さらに、投与は、組成物のボーラスの定期的な注射によるものであり得るかまたは体外（例えば、静脈内バッグ）または体内（例えば、生物分解性の（ｂｉｏｅｒｏｄａｂｌｅ）移植）であるリザーバからの静脈内または腹腔内投与によってより連続的になされ得る。
【００８６】
本発明によって意図される治療的組成物を、任意の適切な手段によって、直接（例えば、注射、移植または組織部位に対する局所的投与によるように局所的に）、または全身的に（例えば、非経口的にまたは経口的に）個体に提供し得る。組成物が非経口的に（例えば、静脈内、皮下、分子内、眼、腹腔内、筋内、頬、直腸、膣、眼窩内、頸部内、頭蓋内、脊髄内、室内、鞘内、大槽内、嚢内、鼻腔内によって）またはエアロゾル投与によって提供される場合、この組成物は、水性または生理学的に適合な液体懸濁物または溶液の部分を含み得る。従って、キャリアまたはビヒクルは、生理学的に受容可能であり、その結果、所望の組成物の被験体への送達に加えて、患者の電解質および／または容量バランスに他の悪影響を及ぼさない。
【００８７】
非経口投与に有用な溶液は、薬学の分野で周知の方法（例えば、ＲＥＭＩＮＧＴＯＮ’Ｓ　ＰＨＡＲＭＡＣＥＵＴＩＣＡＬ　ＳＣＩＥＮＣＥ（Ｇｅｎｎａｒｏ，Ａ．、編）、Ｍａｃｋ　Ｐｕｂ．、１９９０に記載される方法）のいずれかによって調製され得る。本発明の治療用薬剤の処方物としては、例えば、ポリアルキレングリコール（例えば、ポリエチレングリコール、植物起源の油、水素化ナフタレンなど）が挙げられ得る。特に、直接投与のための処方物は、所望の部位での薬剤の維持を補助するグリセロールおよび他の高粘度の組成物を含み得る。生体適合性、好ましくは生体吸収性（ｂｉｏｒｅｓｏｒｂａｂｌｅ）のポリマー（例えば、ヒアルロン酸、コラーゲン、リン酸三カルシウム、ポリブチレート、ラクチドならびにグリコリドポリマーおよびラクチド／グリコリドコポリマーを含む）は、インビボで薬剤の放出を制御する有用な賦形剤であり得る。ペプチド薬物についての徐放性注射可能処方物の概念は、十分受け入れられており、そしていくつかの利点を提供する。第一に、例えば、バイオアベイラビリティーが高い。第二に、処置レジメンは、１ヶ月または３ヶ月当たり１回（ＡｂｏｔｔのＬｅｕｐｒｏｎ（登録商標）のように）または１年に１回（例えば、ＡｌｚａのＶｉａｄｕｒ（登録商標））からなり得る。第三に、徐放性注射可能処方物は、実質的に、使用され得る用量を減少させる（Ｌｅｕｐｒｏｎ注射用量は、１ｍｇ／日であるが、９０日の処方物使用量（ｆｏｒｍｕｌａｔｉｏｎｕｓｅ）は、総量１１．２５ｍｇである）。また、増大した効力は、治療が感染性を予防するために連続して存在する場合に達成され得る。この考えは、感染のｓｌｏｗ−ｔｏ−ｃｌｅａｒ沈着の再形成（例えば、記憶Ｔ細胞コンパートメント）を予防することにより疾患の治癒を達成する必要性の観点から特に重要である。例えば、Ｌｅｅ，Ｖ．編、Ｐｅｐｔｉｄｅ　ａｎｄ　Ｐｒｏｔｅｉｎ　Ｄｒｕｇ　Ｄｅｌｉｖｅｒｙ、Ｍａｒｃｅｌ　Ｄｅｋｋｅｒ，Ｉｎｃ．、ＮＹ（１９９１）を参照のこと。
【００８８】
これらの薬剤のための他の潜在的に有用な非経口送達システムとしては、エチレン−ビニルアセテートコポリマー粒子、浸透圧ポンプ、移植可能な注入システム、およびリポソームが挙げられる。吸引投与のための処方物は、賦形剤として、例えば、ラクトースを含むか、または水溶液（例えば、ポリオキシエチレン−９−ラウリルエーテル、グリココレートおよびデオキシコレートを含む）かもしくは鼻ドロップまたは鼻腔内へ適用されるためのゲルの形態での投与のための油性溶液であり得る。非経口投与のための処方物はまた、頬投与のためのグリココレート、結腸投与のためのメトキシサリチラート、または膣投与のためのクエン酸（ｃｕｔｒｉｃ　ａｃｉｄ）を含み得る。
【００８９】
本発明のさらなる局面および実施形態は、当業者に明らかである。
【図面の簡単な説明】
【図１】図１は、固定された非変性リジンもしくはアルギニン、ならびにおおよそ等しい割合の１８アミノ酸からなる８個の変性位置を有する、ペプチドライブラリーを示す。
【図２】図２は、結合ドメインを用いたペプチドライブラリーのスクリーニングを示す。
【図３】図３は、ライブラリーからのアフィニティー精製のためのＧＰＣＲの固定化を示す。[0001]
(Related application)
This application claims the benefit of US Patent Application Nos. 60 / 190,946, 60 / 190,996 and 60 / 191,299, the disclosure of each of which is incorporated herein by reference. Invite to Reference is also made to applications filed on March 20, 2001, identified by attorney docket numbers CNS-005 and CNS-007, the disclosures of each of which are incorporated herein by reference. .
[0002]
(Field of the Invention)
The present invention relates generally to G protein-coupled receptors ("GPCRs"), and more particularly to compounds that bind to G protein-coupled receptors. The methods of the present invention are useful for treating diseases by identifying and preparing therapeutic compounds that bind to G protein-coupled receptors.
(Background of the Invention)
Receptors are generally molecular structures located in the cell membrane or intracellularly that form weak reversible bonds with extracellular factors such as antigens, hormones or neurotransmitters. Each receptor is designed to bind a specific factor. A particular family of receptors is the seven transmembrane ("7TM"), G-protein coupled receptor ("GPCR"). These receptors are linked to guanine nucleotide binding proteins ("G proteins") in order to transmit signals from extracellular factors to which these receptors bind. When the G protein binds to guanine diphosphate ("GDP"), the G protein is inactive or "off-position." Similarly, when the G protein binds to guanine triphosphate ("GTP"), the G protein is active or "on position," thereby activating an intracellular biological response. Mediated.
[0003]
Generally, the activity of almost all cells in the body is regulated by extracellular signals. Many physiological events in humans and with a wide range of organisms use protein-mediated transmembrane signaling via GPCRs. Signals from certain GPCRs cause activation of G proteins in the cell. Most of the signal is transmitted inside the cell by the GPCR. Signal transduction is achieved by ligand binding and activation of the linked G protein. There are many aspects to this signaling process, including multiple receptor subtypes of GPCRs and their G protein-linked equivalents, as well as various linked intracellular messengers. This signaling can result in total or partial activation or inactivation of the intracellular process (s), depending on the protein involved. Important signaling molecules or neurotransmitters that bind to GPCRs include, for example, morphine, dopamine, histamine, 5-hydroxytryptamine, and adenosine.
[0004]
GPCRs constitute a superfamily of proteins. Currently, over 2000 GPCRs have been reported in the literature, which fall into three families: the rhodopsin-like family, the metabotropic glutamate family, and the calcitonin receptor. For example, Ji TH et al. Biol. Chem. 273 (28): 17299-302 (1998). The reported GPCRs include both characterized receptors and orphan receptors for which no ligand has been identified. See, for example, Wilson S et al., G protein-coupled receptors. Haga T, Bernstein G, eds. CRC press, Boca Raton, 97-116 (1999): Wilson S et al., Br. J. Pharmacol. 125 (7): 1387-92 (1998); and Marchse A et al., Trends Pharmacol. Sci. 20 (9): 370-375 (1999). Generally, despite the large number of GPCRs, each GPCR shares a similar molecular structure. Each GPCR contains a string of amino acid residues of various lengths. GPCRs reside in the cell membrane in seven separate coils called transmembrane. The amino terminus of the GPCR is outside the cell with an extracellular loop, while the carboxyl terminus is inside the cell with an intracellular loop.
[0005]
In general, similarities in tertiary structure are also shared by G proteins as well. Typically, G proteins are also called heterotrimeric G proteins and consist of three subunits, α, β and γ. In a typical G protein, the α subunit contains two domains, a GTPase domain and an α-helical domain. This GTPase domain contains a helix surrounding the β sheet. The α-helix domain is unique to G proteins and includes a long central helix surrounded by five shorter helices. The β and γ subunits are often called β-γ dimers. The β subunit is a “β propeller” protein that includes a sheet, an α helix, and a loop connecting the helix with a “propeller blade”, arranged like a blade on a propeller. On the other hand, the γ subunit generally has no endogenous tertiary structure; instead, structural support is believed to depend on the β subunit. Interactions with the β subunit are believed to be mediated, in part, by coiled coil interactions between the N-terminal helices of the individual subunits.
[0006]
Ligands for GPCRs include small molecules and peptides and small proteins. The interactions between these ligands and their receptors vary from system to system, but they may all require interaction with the four extracellular domains and residues at some of the N-termini. GPCRs with known ligands include multiple sclerosis, diabetes, rheumatoid arthritis, asthma, allergy, inflammatory bowel disease, some cancers, thyroid disease, heart disease, retinitis pigmentosa, obesity, neurological disorders , Osteoporosis, human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome ("AIDS"). See, for example, Murphy PH et al., Pharm. Rev .. 52 (1): 145-176 (2000); Mannstadt M et al., Am. J. Physiol. 277 (5): F665-75 (1999); Berger EA et al., Ann. Rev .. Immunol. 17: 657-700 (1999); Saunders J et al., Drug Discov. Today 4 (2): 80-92 (1999); Hebert TE et al., Biochem. Cell. Biol. 76 (1): 1-11 (1998); Jacobson ED et al., Dig. Dis. 15 (4-5): 207-42 (1997); Meji JT, Mol. Cell. Biochem. 157 (1-2): 31-8 (1996); and Chanmers J et al., Nature 400: 261-4 (1999). In general, G protein-coupled receptors can be further divided into different classes based on the type of ligand they bind: peptides, biogenic amines, nucleotide-related, lipid-based, amino acid-based, and retina (ie, light-based). Of particular interest are, for example, the class of receptors that bind peptide substrates, such as CC chemokine receptor 5 and CXC chemokine receptor 4, which is the largest defined class.
[0007]
Although GPCRs have been shown to serve as receptors for signal transduction, it has been difficult for researchers to obtain high resolution X-ray crystallographic structures of GPCRs. Because of the difficulty in crystallizing 7TM proteins that require complex interactions with lipids for native conformation. The need for interaction with lipids also makes it difficult to prepare biologically active forms of GPCRs. Because, in the absence of these lipids, they have a minimal ability to specifically bind ligands unless great care is taken to maintain a biologically active conformation during solubilization. This is because denatured aggregates, which may or may not be capable, are readily formed. Various approaches have been used to define the regions of the GPCR that are involved in ligand binding in the absence of X-ray structures. These approaches generally involve comparing the results with non-human homologs, chimeric receptors, and point mutants to study the structural requirements for GPCR activity.
[0008]
For example, specific GPCRs associated with HIV infection and AIDS, such as CC chemokine receptor 5 ("CCR5") and CXC chemokine receptor 4 ("CXCR4"), serve as co-receptors for HIV, so that , Interacts with HIV and facilitates virus entry into cells. Accordingly, research has sought to identify natural ligands that act as therapeutic agents that inhibit the entry of the virus into cells. For other GPCRs, CCR5 and CXCR4 are very difficult to solubilize and purify. Because they usually need to be folded and maintained in the presence of cell membrane native lipids.
[0009]
Accordingly, there is a need in the art for methods of identifying GPCR binding compounds and for identifying GPCR binding therapeutics for preventing or treating diseases and disorders such as, for example, HIV infection and AIDS. Such therapeutics can include peptides, peptidomimetics, or small molecules that can inhibit binding of the natural ligand to the GPCR. Such methods and compositions are provided herein.
[0010]
(Summary of the Invention)
The present invention provides compounds that bind to GPCRs and methods for identifying these binding compounds. In one embodiment, a screening method for providing a motif that binds to a GPCR and a ligand that can bind to the GPCR is provided. In another embodiment, the invention includes the design and identification of therapeutic peptides, peptidomimetics, or small molecules suitable for use in the prevention or treatment of diseases or disorders, such as, for example, HIV infection and AIDS.
[0011]
In one embodiment, the methods of the invention provide for the synthesis and purification of linear and cyclic peptide libraries useful for screening and identifying motifs that bind to GPCRs and for screening for their potential ligands. The methods of the present invention provide for the incorporation of unnatural amino acids and amino acids in the D conformation into linear or cyclic peptides for use in such libraries. Libraries containing peptides with such amino acids exhibit an increased in vivo binding affinity and duration of action in vivo resulting from resistance to proteolysis.
[0012]
In a preferred embodiment, the present invention provides for the use of a highly diverse library of peptides (linear and cyclic, natural and unnatural amino acids), peptidomimetics, and small molecules for the lead ligand identification step. I do. Such a ligand can be an agonist or antagonist, directly or indirectly, for GPCR binding activity.
[0013]
In a preferred embodiment, the present invention provides for the use of a phage display method for the identification of preliminary motif information, followed by the additional use of the purified receptor preparations of the present invention and affinity purification using a highly diverse library. Provide a round. In a particularly preferred embodiment, phage display technology is combined with the use of a cyclic peptide and / or peptidomimetic library.
[0014]
In another embodiment of the invention, the lead compound is substantially screened, identified, designed, or evaluated for agonist or antagonist capacity for GPCR activity using computer-aided design techniques. Such techniques use computer-generated three-dimensional images by actually aligning the protein with the binding partner based on the molecular and structural information of both the GPCR and the potential binding partner. In the case of libraries designed for computer-aided screening, the large amount of information required for lead optimization is obtained directly from library design. In one embodiment, potential reads are identified by pre-screening the actual library or by some other means. One embodiment of the present invention involves biologically relevant and drug screening by rational structure-based drug design. In such cases, the three-dimensional structure of the protein (or similar family member), peptide or molecule is determined, and potential agonists and / or antagonists are designed with the aid of computer modeling. In a preferred embodiment of the invention, after a suitable drug has been identified, the drug is contacted with a GPCR, whereby a binding complex is formed between the potential drug and the GPCR. Methods for contacting a drug with a GPCR are generally understood by those skilled in the art of drug development.
[0015]
In another embodiment, the present invention relates to the use of partially purified GPCRs as an agent to perform the selection, identification, and improvement of tightly bound ligands in identifying therapeutically useful compounds. provide. In a preferred embodiment, the present invention involves the use of a labeling method to generate a modified GPCR, wherein the protein functions to facilitate the purification and identification steps involved in the screening method. In another embodiment, the invention relates to a tag sequence having an appropriate specific protease site engineered in a vector (ie, GST, FLAG, 6 × His, double-labeled FLAG-GST, C-MYC, MBP, V5, Xpress, CBP, HA).
[0016]
In a particularly preferred embodiment, the methods of the present invention provide for solubilization and immobilization of GPCRs, facilitating the ligand selection methods provided herein. GPCRs can be from any source, including but not limited to inactive precipitated protein preparations; cell membrane preparations; and whole cell preparations. In one embodiment, the invention provides a method of directly screening combinatorial libraries for general affinity determination using membranes from a baculovirus expression system or any other suitable expression system. In one embodiment of the present invention, partially purified GPCRs are used to select, identify, and improve tight binding ligands. In a preferred embodiment, partially purified labeled GPCRs are used in isolated form and screened for affinity purification of ligands that tightly bind diversified libraries (centralized or highly diversified) Is done. In a preferred embodiment of the invention, the conditions for solubilization and immobilization of the appropriate ligand are low salt (eg, low magnesium or calcium concentration); and no sodium chloride (“NaCl”) (0.0 nM NaCl). ) To provide for use.
[0017]
In another embodiment, the invention involves eluting the binding components of the library from immobilized proteins with peptides or N-terminal specifically blocked peptides or other analogs that cannot be sequenced. In yet another embodiment, the invention includes the step of binding the combinatorial library to a resin-immobilized protein. In another embodiment, the invention includes a purified polypeptide having a labeling sequence, which polypeptide may be immobilized on an affinity resin suitable for the assay. A further embodiment comprises the step of releasing or eluting the labeled protein with its binding library using a peptide whose N-terminus is specifically blocked or other non-sequenceable analog. In yet another embodiment, the method of the present invention comprises removing a label from the protein of interest (protein / protein) after being immobilized on the affinity resin and after the combinatorial library is bound to release the complex. Cutting with a specific protease (as designed in the vector).
[0018]
In yet another embodiment, the targeting ligand is a library of linear peptides, a library of peptidomimetics, a library of cyclic peptides, or a focus developed using the initial motifs identified by phage display technology. The library is selected from a focused library or a library combining any of the above. In another embodiment, the targeting ligand may be a peptide or other ligand, or a pH-change or chaotropic agent such as urea or guanidine hydrochloride, which may influence the hydrogen bonding structure and hydrophobicity of water. (Which can denature proteins in concentrated solutions by reduction) is eluted from the receptor preparation. Ligands to GPCRs identified using the methods disclosed herein are also contemplated by the present invention. In yet another embodiment of the present invention, protein sequencing techniques are used for the structure determination of the ligand identified by the affinity purification step.
[0019]
In another embodiment, the invention is directed to a small molecule antagonist of GPCR binding identified using the methods of the invention, eg, suitable for treating a disease or disorder (eg, such as an HIV infection or AIDS). Various therapeutic agents. In another embodiment, a patient suffering from a disease or disorder is treated with a therapeutic agent comprising a compound identified using a method of the invention (eg, such as small molecule antagonist binding of a GPCR). In another embodiment, patients suffering from a disease or disorder (eg, such as HIV infection and AIDS) are treated through the combined use of therapeutic agents (eg, including GPCR inhibitors and reverse transcriptase and protease inhibitors). You.
[0020]
A detailed description of certain preferred embodiments of the invention is provided below. Other embodiments of the invention will be apparent from a review of the ensuing detailed description.
[0021]
(Detailed description of the invention)
In general, the methods of the invention provide for determining the binding motif of a GPCR. Further, the methods of the present invention provide for the identification of agonists or antagonists of the interaction of a GPCR with its natural ligand, thereby providing the identification of a therapeutic lead compound. Methods for library design and library synthesis, and methods for library screening, which are particularly useful in the present invention, are described in the following patents and patent applications, the disclosure of each of which: Are incorporated herein by reference: Cantley et al., US Patent No. 5,532,167; Cantley et al. S. S. N. 08 / 369,643 (filed December 17, 1998); Cantley et al. S. S. N. 08 / 438,673 (filed Nov. 12, 1999); Hung-Sen et al., U.S. Pat. S. S. N. 09 / 086,371 (filed May 28, 1998); Hung-Sen et al. S. S. N. 08 / 864,392 (filed June 24, 1999); and Lai et al. S. S. N. 09 / 387,590 (filed on August 31, 1999).
[0022]
According to the methods of the present invention, GPCRs are cloned and expressed and tested for activity. GPCRs can be labeled on the C-terminus or on the N-terminus to facilitate characterization of the ligand binding properties of the GPCR. Exemplary labels include, but are not limited to, 6 × His, FLAG, GST, V5, Xpress, c-myc, HA, CBD, and MBP. Labeled GPCRs are used, for example, in screening libraries containing linear and / or cyclic peptides, having natural and / or non-natural amino acids, peptidomimetics and / or small molecules. Such peptidomimetics and small molecules can be used to screen any natural or synthetic compound, composition, chemical, protein, or GPCR binding compound for any of the above or any combination of GPCRs. Any of the above modifications used in screening for binding compounds may be included.
[0023]
In one aspect, an oriented modified peptide library method useful in the methods of the invention comprises one or more amino acids in a non-denatured position known or suspected to be important for ligand binding; For example, a soluble peptide library of approximately equal proportions of 18 amino acids at the denatured position is used. Cysteine and tryptophan can be deleted to avoid certain analytical difficulties for sequencing. Such a library is shown in FIG. 1, where X represents a denatured position consisting of any 18 amino acids and lysine or arginine is fixed at the non-denatured position. Additional residues can be added to the N-terminus of the sequence shown in FIG. This is because interfering substances are often present in the first and second sequencing cycles. Additional residues can be added at the C-terminus to provide amino acids for better anchoring the polypeptide to the filter in the sequencer cartridge.
[0024]
Another aspect of the invention is the use of a very diverse library of peptides (linear and cyclic amino acids, natural and unnatural amino acids), peptidomimetics, and small molecules for subsequent identification steps I will provide a. For example, these ligands can be repulsive or antagonistic in this function for the receptor. In general, the present invention uses partially purified GPCRs as a factor to perform the selection, identification, and improvement of tightly bound ligands as a pathway for therapeutically useful compounds. Further, the invention provides for the development and use of solubilization and immobilization procedures that facilitate the efficient ligand selection methods provided herein. In particular, optimal conditions for efficient ligand selection for a particular GPCR include the use of low salts (eg, low or no magnesium or calcium concentration and no NaCl concentration. Further provided herein are methods for ligand selection using inactive precipitated proteins, cell membrane preparations and whole cell preparations.
[0025]
In one aspect of the invention, the screening step may include phage display technology. Such phage display systems are used to screen peptide libraries that bind to selected target molecules, and to display functional proteins that have the potential to screen these proteins for desired properties. Has been used. More recent improvements in this display approach allow the expression of enzymes as well as antibody fragments on the bacteriophage surface, thus allowing selection of particular properties by selecting with particular ligands. See, for example, Smith SF et al., Methods Enzym. 217: 228-257 (1993). The phage display method has been used for the identification of preliminary motif information and has subsequently been used to purify the purified receptor preparations of the present invention and a very diverse library, particularly libraries of cyclic peptides and peptidomimetics. It can be used for additional rounds of affinity purification used. This phage display method allows to identify natural amino acid motifs. Information from phage display can be incorporated into affinity purification methods using synthetic libraries containing novel amino acid analogs or cyclic peptides, for example, to select ligands with enhanced pharmaceutical properties. The use of initial, secondary and tertiary libraries makes the definition of binding site specificity more complete. Secondary libraries that incorporate information from the initial library can be sequenced. In the initial library, some degenerate positions may yield high performance for certain amino acids, and these may be non-denatured positions consisting of preferred amino acids in the second library. See, for example, Wu R, J Biol Chem 271 (27): 15934-41 (1996).
[0026]
Alternatively or additionally, computer-aided design techniques may be used in screening and / or designing peptides, peptidomimetics and small molecules. For example, in addition to GPCR sequences, transmembrane prediction, and any structural information obtained from mutagenesis studies, see the crystal structure of rhodopsin (eg, Palczewski et al., Science 289 (5480): 739-745 (2000)). With such information, computer-aided design techniques can, in effect, screen, identify, design and test potential compounds for their GPCR activity. Computer programs that can be used to aid in the design of appropriate peptides, peptidomimetics and small molecules include, for example, Dock, Frodo and Insight. Examples of methods for screening biologically relevant drugs rely on structures based on rational drug design. In such cases, the three-dimensional structure of the protein, peptide or molecule is determined or modeled, and potential agonists and / or antagonists are designed using computer modeling. See, e.g., Butt et al., Scientific American, December 92-98 (1993); West et al., TIPS, 16: 67-74 (1995); Dunbrook et al., Folding & Design, 2: 27-42 (1997). After the appropriate drug has been identified, the drug is contacted with the GPCR, where a binding complex is formed between the potential drug and the GPCR. Methods of contacting a GPCR with a drug are generally understood by those skilled in the art of drug development.
[0027]
The screening step may be performed in the liquid phase or using a GPCR immobilized on an affinity column. In addition to immobilizing labeled GPCRs using an affinity resin, other forms of sequencing can be used to perform affinity purification of a selected ligand from a library. These embodiments include, but are not limited to, the following examples. The receptor and bound library components can be separated from unbound library components using equilibrium dialysis. The labeled receptor can be bound to a specific affinity membrane, which is in the form of a plate or separate. The library can then be incubated with the membrane and easily washed to remove non-specific binding components. Size exclusion methodology can be used to separate the purified receptor-bound library complex from unbound components after pre-incubation of the receptor with the library. In addition, the micelle complex containing the receptor, which may or may not incorporate lipids and detergents, is separated from the library by differential centrifugation after binding of the selective affinity component. obtain. In general, high affinity ligands can be released using low pH or high salt concentrations, and the structure is identified by sequencing described herein.
[0028]
In order to determine those ligands with the highest affinity for the target receptor, generally more than 200 peptide libraries can be screened to determine the respective inhibitory binding of each library. In general, at 100 μM, greater than 10% inhibition was significant for subsequent evaluation of the sequence via affinity purification. In a further aspect of the invention, once a preferred amino acid residue has been identified by a GPCR at a degenerate position in the library due to a high preference value, the particular peptide is required for library synthesis. Synthesized by the same method used. In one embodiment of the invention, the high priority value is greater than one. This value is determined by subtracting the control value from the sample value and dividing by the reference value. In a preferred embodiment of the invention, this priority value is greater than 1.2. In a more preferred embodiment of the present invention, this priority value is greater than two. After synthesis of the identified peptide sequence, the peptide is purified, for example, by high performance liquid chromatography ("HPLC"), and the composition is determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry ("MALDI-TOF MS"). ) And Edman sequencing. In general, relative affinity can be measured by modifying a radiolabel binding assay used in receptor purification.
[0029]
Other methods can be used to enhance the specificity of the motif obtained from the affinity purified peptide. The binding components of the library can be eluted from the immobilized protein with a particular N-terminal block peptide or other non-sequenced analog. To avoid release of trace contaminants from the affinity resin after library binding, the release / elution of GPCRs labeled with that binding library may require specific N-terminal block peptides or other non-sequenceable This can be achieved using analogs. This is done using an acetylated FLAG peptide, which can elute the GPCR-FLAG receptor from the resin. Alternatively, the GPCR-derived label can be immobilized on an affinity resin and after the combinatorial library is released from the complex, after a specific protease (such as designed in a protein / vector; enterokinase or Thrombin). Finally, the libraries can be tested for their ability to bind to receptors (using significantly less protein) in single or array assays, eg, Sf9 cells (Spodoptera frugiperda) or High Five cells (Trichoplusia). ni) can be prescreened by a binding assay using a GPCR-containing membrane from (both available from Invitrogen). This screen can be performed using GPCRs and many linear and circular libraries to determine the effectiveness of these libraries in inhibiting the natural ligand for binding.
[0030]
In addition, other receptors and soluble proteins, in addition to GPCRs, can be screened using this method to produce agonists or antagonists for therapeutic use. Examples of types of proteins according to this screening methodology include single transmembrane proteins, PIG tail receptors, progesterone receptors, arrestins, nuclear receptors, cytokine receptors, and essentially any protein binding protein, or any peptide binding Including but not limited to proteins. IL-12 is one type of cytokine receptor, which is useful in the methods of the invention to identify therapeutic compounds for the treatment of diseases such as type I diabetes. See, for example, Falcone M et al., Curr. Opin. Immunol. 11: 670-676 (1999). PIG tail receptors include cathepsin D (25) and natural killer cell receptors (eg, CD48 and CD55) (26). See, for example, Ogier-Denis E et al., Biochem. Biophys. Res. Comm. 211 (3): 935-42 (1995); and Scubert J et al., Blood 76 (6): 1181-7 (1990). Progesterone receptors are well-known nuclear receptors with biological relevance. See, for example, Chauchereau A et al. Biol. Chem. 275 (12): 8540-8548 (2000); Fewings PE et al. Neurosurg. 92 (3): 401-405 (2000). Arrestin is a family of soluble protein binding proteins that are involved in a wide range of diseases by their role in signal termination. See, for example, Wilson CJ et al., Curr. Biol. 3 (10): 683-6 (1993). These are just a few examples of proteins that can be easily applied to this format.
[0031]
The methods of the present invention further include peptides, peptidomimetics, and / or small molecules that are antagonistic to GPCR activity suitable for treating a patient having a disease or disorder such as HIV infection or AIDS. Including therapeutic agent design. Identification of binding compounds for GPCRs and optimal synthesis and their purification provide an effective treatment for many diseases or disorders, such as AIDS and HIV infection. In general, the small peptide ligand binding compounds of the invention (both cyclic and linear peptide ligands) have increased binding affinity and potency, and show resistance to proteolysis. For example, CXCR4, CCR5, gonadotropin-releasing hormone receptor ("GnRHR"), and peptides from libraries for certain cyclic peptides have been found to exhibit increased binding activity to target proteins.
[0032]
Particular embodiments of the present invention are described in the following examples, which are not meant to be limiting.
[0033]
(Example)
Example 1 Preparation of Labeled GPCRs and Screening Using Oriented Linear Peptide Libraries
A variety of GPCR vectors can be prepared for baculovirus expression systems containing epitope tags using standard techniques known to those of skill in the art, which will make receptor purification easier. Labels can be incorporated at the N-terminus or C-terminus of the protein. For GPCRs, the label at the C-terminus of the receptor is incorporated to determine the ligand binding properties of the receptor, which is at the N-terminus or extracellular region of the molecule. The construction of the C-terminal 6 × His tag construct and the C-terminal FLAG construct are provided below as examples. Alternative labels can include, for example, GST, V5, Xpress, c-myc, HA, CBD, and MBP. These constructs were made by analogous procedures using standard techniques known to those skilled in the art.
[0034]
This 6 × His tag allows for one-step purification using nickel chelation. CDNA for a GPCR can be isolated from an appropriate cDNA library using the polymerase chain reaction ("PCR") and primers for the 3 'and 5' ends of the desired gene, and the center of this gene. . To prepare a C-terminal label, the gene of interest was The E. coli vector (pET30a) was subcloned using a C-terminal 6 × His tag. The newly created receptor was then excised and ligated into pBlueBac (Baculovirus transfer vector (Invitrogen, Carlsbad, CA)). This construct is analyzed using both restriction digestion and sequencing, and then Sf9 insect cells (Pharmingen, San Diego, CA) for expression as typically performed by one skilled in the art of protein expression. Transfected.
[0035]
A C-terminal bacterial FLAG construct is available from Sigma (pFLAG-CTC). A similar strategy using standard techniques was used for the construction of this vector. The GPCR was ligated with the pFLAG-CTC plasmid, then subcloned into a C-terminal FLAG tag and ligated into the digested pBlueBac vector. This construct was analyzed using both restriction digestion and sequencing and transfected into Sf9 or High Five insect cells for expression.
[0036]
To express the GPCR gene in Sf9 cells or High Five cells, the pBlueBac vector containing the GPCR insert was co-expressed with Bac-N-Blue DNA using cationic liposome-mediated transfection using standard techniques. Transfected. The GPCR was inserted into the baculovirus genome by homologous recombination. Cells were monitored from 24 hours to 4-5 days after transfection. After about 72 hours, the transfection supernatant was assayed for recombinant plaques using a standard plaque assay. Cells carrying the recombinant virus produced blue plaques when grown in the presence of X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside). The plaques were purified and the isolate was confirmed by PCR for recombination accuracy using standard techniques. This generated a high titer stock and infection was performed from this stock using standard techniques for expression studies. Controls for transfection include cells only and transfer vectors.
[0037]
Constructs for the expression of GPCRs are made from the starting vector pBlueBac4.5 (Invitrogen), which can remove thrombin and enterokinase cleavage sites in the vector. The GST tag is added to the multiple cloning site using PCR to generate a GST tag, which is then ligated to the cut vector (SmaI / EcoRI) using standard procedures known to those skilled in the art. The vector is then made compatible using Gateway technology provided by Lifetech for ease of operation. This is achieved by ligation to a SmaI site cassette containing the desired recombination sites for this technique (obtained from Lifetech). The GPCR of interest is amplified using PCR with primers to extend the gene to include an adhesion site for recombination. This PCR product was then incorporated into a baculovirus vector using BP clonase (enzyme required for homologous recombination), and without the enterotoxin or thrombin cleavage site for baculovirus expression, the C A vector containing a GPCR having a terminal GST tag is prepared. This vector is co-transfected into Sf9 cells for preparation of the viral stock required for expression. The virus is a purified plaque, and the PCR and sequenced clones can be used for expression of a GPCR. The time course of this construct with three GPCRs demonstrated that less time was required for maximal expression of the receptor and less proteolysis. For example, CCR5, CXCR4, and GnRHR required 24-48 hours for expression. Less proteolysis occurred in GnRHR and CCR5.
[0038]
To express a GPCR-labeled receptor in Sf9 cells or High Five cells, a pBlueBac vector containing a labeled insert was combined with Bac-N-Blue DNA using cationic liposome-mediated transfection using standard techniques. Co-transfected. This labeled receptor was inserted into the baculovirus genome by homologous recombination. Cells were monitored from 24 hours to 4-5 days after transfection. After approximately 24-72 hours, the transfection supernatant was assayed for recombinant plaques using a standard plaque assay. Cells carrying the recombinant virus produced blue plaques when grown in the presence of X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside). The plaques were purified and the isolate was confirmed by PCR for recombination accuracy using standard techniques. This generated a high titer stock and infection was performed from this stock using standard techniques for expression studies. Controls for transfection include cells and transfer vectors.
[0039]
Sf9 cells or High Five cells were maintained as both adherent and suspension cultures using standard techniques known to those skilled in the art. The adherent cells were grown to confluence and passaged at a ratio of 1: 5 using sloughing technique. The suspended cells were placed in a spinner flask with 0.1% pluronic F-68 (to minimize shear) at 1 × 10 ⁶ Maintained for 2-3 months by subculturing to a density of cells / ml.
[0040]
The time course following infection with the recombinant virus was used to determine optimal growth conditions for expression using standard techniques. Aliquots of cells from spinner flasks were taken over this time course, centrifuged at 800 × g for 10 minutes at 4 ° C., and both supernatants and pellets were assayed by SDS-PAGE / Western blot analysis. This GPCR was predicted to be in the membrane fraction (pellet). All viable systems were assayed in this manner for levels of expression. A standard radiobinding assay is performed with the natural ligand in the membrane preparation to evaluate the system for activity. Where the natural ligand is not known, as is often the case with some orphan receptors (where the activity of the receptor is not defined), this activity can be determined using standard cell-based assays known to those skilled in the art. And assayed for G protein-coupled signal activity.
[0041]
The membrane fraction is isolated by first pelleting all Sf9 cells (800 × g, 10 minutes at 4 ° C.) and then resuspending the pellet in lysis buffer by homogenization. Representative lysis buffers are near neutral pH and include a cocktail of protease inhibitors, both of which are standard techniques for those skilled in the art. For example, serine proteases, cysteine proteases, aspartyl proteases, and metalloproteases can be used with inhibitors such as PMSF, aprotinin, leupeptin, phenatroline, benzamidine HCl. Pellet the membrane. Solubilization can also be performed by using different surfactants (eg, β-dodecyl maltoside, n-octyl-glucoside, CHAPS, deoxycholic acid, NP-40, Triton X-100, Tween-20, digitonin, amphoteric detergent, CYMAL, Solubilization of this receptor by, but not limited to, lauroyl sarcosine, was compared for quantity and activity. Solubilization was also performed using varying NaCl concentrations. Despite conventional beliefs, this step of solubilization using low salts (eg, low calcium and magnesium concentrations) and substantially free of NaCl, was unexpected when compared for quantity and activity. Optimum solubilization conditions were obtained. Having 0.0 nM NaCl provided the best conditions for counter-intuitive but solubilizing and immobilizing candidates with binding properties (eg, CCR5 and CXCR4). Candidates for isolation achieved purification as described below.
[0042]
After determining the appropriate detergent (eg, NP-40) for solubilization and activity, GPCRs were purified from membrane fractions. The exact purification scheme will depend on the construct chosen, which is a matter of activity and ease of solubilization. For purification of the 6 × His-labeled receptor, the membrane fraction was loaded onto a Ni-NTA column (Quiagen, Valencia, Calif.) In the presence of a detergent (eg, NP-40) and washed thoroughly. And eluted with imidazole. Purification of the FLAG-labeled receptor was performed using an anti-FLAG M2 affinity matrix (Sigma, St. Louis, MO) in the presence of detergent and eluted with glycine. This purification was performed in the presence of a suitable detergent (eg, NP-40) found for the experimental system described herein. The activity of the purified receptor was assessed as described herein.
[0043]
The peptide was converted to 9-fluorenylmethyloxycarbonyl / ter. -Mounted on a Rink amide resin (NovaBiochem, substitution level 0 / .54 mmol / g) via a method based on -butyl ("Fmoc" / "tBu") using an Applied Biosystems 433A synthesizer. tBu was used for protection of the side chains of Asp, Glu, Ser, Thr and Tyr, and tert. -Butyloxycarbonyl ("Boc") is used for protection of the side chains of Lys and Trp, and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl ("Pbf") is Used for side chain protection, and triphenylmethyl ("trityl", "Trt") was used for Cys, His, Asn and Gln side chain protection. The scale of the synthesis was 0.20 mmol. The resin was first washed with N-methylpyrrolidinone ("NMP") and then treated with piperidine: NMP (1: 4) for 1 × 3 minutes and 1 × 7.6 minutes with N ^α -Performed for Fmoc removal. All Fmoc-amino acids were converted to N-[(1H-benzotriazol-1-yl) (dimethylamino) methylene] -N-methylmethaminium hexafluorophosphate N-oxide (N-oxide) according to the following manufacturer's protocol. [(1H-benzotriazol-1-ly) (dimethylamino) methylene])-N-methylmethanaminium hexafluorophosphate N-oxide ("HBTU") coupled: (a) Derivatized amino acid of 1.0 mmol of 2.1 mmol of 1.0 mmol of (HBTU) Dissolved in NMP; (b) 0.9 mmol of 0.5 M HBTU in N, N-dimethylformamide ("DMF") was added to the amino acid cartridge and the solution was mixed for 6 minutes. ; ( ) 1.0 mL of NMP containing 2.0 M N, N-diisopropylethylamine ("DIEA") was added to the cartridge; (d) Transfer the HBTU solution to the resin and allow to mix for 40 minutes at room temperature while mixing. Reacted. The resin was filtered and rinsed six times with a total volume of 90 ml NMP and the cycle was repeated. In this one pot method for constructing a highly degenerate and directed peptide library, a batch of resin is reacted with a mixture of combinatorial amino acids without any resolution of the resin. Was.
[0044]
By adjusting the amino acid concentration in the starting mixture, the relative binding rate is controlled, thereby ensuring equal uptake of the amino acids in the library. Optimization of natural Boc-protected amino acids and mixtures of Fmoc-protected amino acids for one-pot synthesis has been described previously (eg, US Pat. No. 5,225,533; Ivanetic et al., Combinatorial). Chemistry, 267 Volume, Academic Press, San Diego, CA USA, pp. 247~260 (1996); Buettner et al., Innovation and Perspectives in Solid Phase Synthesis: Peptides, Proteins, andNucleic Acids, Mayflower Worldwide Ltd., Birmingham, UK, 169 -174 (1994); Ostresh et al., Biopolymers. See 147-155 (1996)): and Herman et al., Molecular Diversity 2; 87~98 (1998):;: 4 1681~9 (1994) Songyang et al, Methods in Mol Biol 87. The cleavage reaction is performed using trifluoroacetic acid ("TFA"): H ₂ O: anisole: triisopropylsilane (“IP ₃ SiH ") (87.5: 5: 5: 2.5, ca. 6 mL) was performed by stirring for 3 hours at 25 ° C. (see, eg, Herman et al., 1996). The filtrate was collected and the resin was further washed with TFA. Cold (−78 ° C.) diethyl ether was added to the combined extracts and the solution was cooled to −78 ° C. After removing the supernatant, the resulting precipitate was washed several times with cold ether, dissolved in glacial acetic acid and lyophilized.
[0045]
For the cyclic peptide library, Fmoc-Asp (OH) -ODmab (Dmab, 4- [N- (1- (4,4-dimethyl-2,6-dioxooxycyclohexylidine) -3-methylbutyl)) Amino] -benzyl (4- [N- (1- (4,4-dimethyl-2,6-dioxycyclohexylidene) -3-methylbutyl) amino] -benzyl) is obtained after the chain is extended as described above. Side chain anchored to Rink amide resin. After linear assembly, removal of the Dmab and Fmoc groups was performed by treatment with hydrazine: DMF (1:49) for 7 minutes and piperidine: NMP (1: 4) for 6 × 3 minutes, respectively. Achieved by The resin was transferred to a syringe containing a polypropylene frit for manual cyclization. Head-to-tail cyclization on resin was performed using 7-azabenzotriazo in a solution containing 1% Trition X in NMP: DMF: dichloromethane, methylene chloride, DCM (1: 1: 1). Performed at 55 ° C. for 2 hours using -l-1-yloxy) -tris (pyrrolidino) phosphonium hexafluorophosphate (“PyAOP”): DIEA (1: 2, 4 equivalents). Unreacted linear precursor was purified with Fmoc-Nva-OH / PyAOP / DIEA (“Nva”, “Norvaline”) (1: 1: 2, 4 eq) in DMF for 1 × 18 hours and 1 × Treated for 3 hours. Subsequent cleavage and side chain deprotection as described above yielded a mixture containing the cyclic peptide library and the corresponding linear (non-cyclized) sequence. The desired cyclic peptide library was purified to remove linear contaminants by reverse-phase high performance liquid chromatography ("RP-HPLC").
[0046]
Peptides and peptide libraries were characterized by HPLC, MALDI-TOF MS (Louisiana State University), and Edman degradation. MALDI-TOF MS analysis allows detection of the presence of high molecular weight impurities due to incomplete deprotection, deblocking, or alkylation. Edman degradation provides quantitative information about the amount of each amino acid at each degenerate position in the library.
[0047]
The synthesized starting library contains a single non-degenerate directed amino acid (i.e., MXXXXXXR-X-X-X-A, where X is other than cysteine). Which is a degenerate equimolar mixture of all amino acids). Circular libraries (head-to-tail) were also prepared with a single, non-degenerate, directed amino acid. Through the use of these starting libraries, optimized residues are defined at some degenerate positions, and a secondary library is created and these positions are fixed. For example, the head-to-tail cyclization library cyclo (M-X-X-X-R-X-X-X-X-N) has a lysine at position -4 (from a fixed R). Should be aspartic acid, position -1 should be histidine, and position +3 should be lysine, so that the second library is It is cyclo (MKXDHHRXXXN).
[0048]
The directed linear peptide library was applied to a column containing an immobilized GPCR and a small fraction of the isolated high affinity peptide. A conceptual diagram illustrating peptide library screening using a binding domain can be shown in FIG. After washing, bound peptide was eluted from the column. Next, both the bound peptide and the entire library applied to this column were separately subjected to Edman degradation and the amino acid distribution as a function of position was determined. Finally, the tendency of the amino acid at the degenerate position is determined. For example, if serine is 5% at position +1 of the amino acid in the starting library, but at position +1 in the high affinity peptide and 15% of the amino acid, position +1 has a preference for serine. I do. The priority value 3 at that position is obtained. Table 1 provides a selective overview of the use of peptide library methods using binding domains.
[0049]
[Table 1]

(Example 2: Preparation and screening of GST-tagged CCR5)
The library was synthesized on ABI 433A (Applied Biosystems, Forster City, CA) using Rink Amide MBHA resin (substitution: 0.54 mmol / gm) with 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. . Amino acid amounts were adjusted empirically in view of literature values to obtain approximately equal coupling of amino acids to degenerate positions. For example, Hook W. et al. Et al., Fed. Proc. 44: 1323 (1985). An exemplary coupling reagent was one equivalent of HBTU / HOBT / DIEA for one equivalent of peptide. Cleavage was achieved with a cocktail (82% TFA, 5% phenol, 5% thioanisole, 2.5% 1,2-ethanedithiol, 5% water). The peptide was precipitated from methyl-tert-butyl ether. The library was characterized by MALDI-TOF MS (Lousiana State University) and amino acid sequencing.
[0050]
The starting library used, for example, a single non-degenerate directed amino acid (i.e., XXXXXXRXXXXX). Secondary libraries can be prepared by immobilizing the optimal residues found at several degenerate positions. For example, XXXXX-R-X-X-X-X should have proline at position -4, so that the secondary library is a P-X-X-X-R -XX-XX.
[0051]
Provided herein is the use of a peptide library with the GST form of the receptor. In the case of a GST-tagged GPCR, the receptor could be exposed to the library and separation of free and bound peptides was achieved by pelleting the membranes by centrifugation. GST-tagged purified receptor was incubated with the peptide library, about 1 μmole of peptide and about 1 nmol of binding sites. After incubation, the receptor with bound peptide was separated from unbound peptide by centrifugation (receptor peptide complex in pellet, unbound peptide in supernatant). Non-specifically bound peptides could be removed by extensive washing, and low pH (less than 2.5) pellet resuspension was used to remove bound peptides. The peptide was sequenced and the consensus sequence was determined.
[0052]
When FLAG-tagged GPCRs were used for peptide library screening, the receptor was immobilized on an anti-FLAG M2 affinity matrix (St. Louis, MO). Further purification approaches could use GST constructs and immobilized glutathione (Pierce, Rockford, IL).
[0053]
Both the bound peptide mixture and the starting peptide library could be sequenced using standard techniques. The amount of each amino acid was determined as a function of position. The preference value for each amino acid at each position was calculated by comparing the amount of amino acids present in the starting live library with the bound fraction of the peptide. Using these procedures, a preferred sequence of peptides that interacted with a number of binding domains was generated and is described in Table 1.
[0054]
Also, a secondary library could be sequenced by incorporating information from the starting library. For this first round of characterization, phage display technology is also used to identify preliminary binding motifs. Phage display provides for the identification of natural amino acid motifs. Phage display technology involves inserting a DNA sequence into a gene encoding one of the phage coat proteins. This gene is inserted at a specific location, and the resulting protein insert can interact with other molecules. As a result, the encoded peptide or protein sequence is displayed on the phage surface and exposed for binding. By inserting degenerate nucleotides, each phage can express a different peptide sequence ("phage library"). Incubation of the phage library with an immobilized receptor can be used to identify sequences that specifically bind to the receptor. Since the signal can be amplified by growing the isolated phage, even weak signals can be detected. Information derived from phage display is applicable to affinity purification methods using synthetic libraries containing novel amino acid analogs or cyclic peptides to select ligands with enhanced pharmaceutical characteristics. The use of starting, secondary and tertiary libraries provided a complete definition of binding site specificity.
[0055]
Once preferred amino acid residues have been identified using high preference values at the degenerate positions of the library, specific peptides can be synthesized by methods similar to those used for library synthesis. These were purified by HPLC and the composition was confirmed by MALDI-TOF MS.
[0056]
Relative affinity can be measured by modifying the iodination binding assay used for receptor purification. Therefore, from this purified receptor membrane [ ¹²⁵ The ability of these peptides to present I] -ligand could be determined. However, if the natural ligand is not known, the activity can be measured using cell-based assays standard in the art. Also, the association constant can be determined using techniques standard in the art.
[0057]
(Example 3: Preparation and analysis of tagged CCR5 and library screening thereof)
(A. Cloning and expression of CCR5)
CCR5 cDNA (see sequence in FIG. 4) was obtained from Receptor Biology (Bertsville, MD) in vector pCDNA3. A tag was added to the C-terminus of the receptor and used to immobilize the receptor for affinity purification assays using standard techniques, as described herein. In addition, a CCR5 cDNA that correlates to GenBank accession number U57840 may be used in the vector pCDNA3. The only difference between the CCR5 cDNA from the Receptor Biology and the CCR5 cDNA that correlates to GenBank accession number U57840 is a point mutation at base number 180 (in this amino acid sequence, changing leucine to glutamine). The following is a specific example from an experiment using this tagging method:
(Construction of CCR5 with C-terminal histidine tag (insect selective expression system))
The CCR5-HIS construct was derived from this system. PCR was performed using CCR5 / pcDNA5 (from Receptor Biology) as a template and using primers 5-Age His and 5-Spe His. With this first primer, a unique SpeI site was induced just before the start ATG of CCR5. The second primer mutated the CCR5 stop codon into the AgeI site, which is in-frame with the histidine tag of pIZT / V5-His (Invitrogen, Carlsbad, CA). The PCR product was digested with SpeI and AgeI and then ligated into similarly digested pIZT / V5-His. This construct is identified as CCR5-His-PIZT.
[0058]
(Construction of CCR5 with C-terminal histidine tag (baculovirus expression system))
CCR5-His-PIZT was digested with HaeII and SpeI and then filled in with Klenow fragment. This fragment containing CCR5-His was ligated into pBluebac 4.5 (Invitrogen) that had been previously digested with Nhel and blunted with Klenow. This construct was checked for directional accuracy. This is called CCR5-HIS. This construct was confirmed by restriction digestion and sequencing using standard techniques. This construct was used for expression and was determined to be well expressed and in an active form for use in affinity purification screening.
[0059]
(Construction of CCR5 having C-terminal FLAG tag)
PCR using standard techniques was performed using CCR5 / pcDNA3 (Receptor Biology) as template and primers 5-Xho pFLAG and primer 5-Sal pFLAG. The first primer engineered a unique XhoI site just before the initiator ATG of CCR5. The second primer mutated the stop codon of CCR5 to a SalI restriction site (in-frame using the FLAG tag of pFLAG-CTC from Sigma). The PCR product was digested with XhoI and SalI and ligated into similarly digested pFLAG-CTC (a bacterial expression vector). This construct is called CCR5-FLAG-CTC. CCR5-FLAG was then digested with XhoI and ScaI and filled with Klenow fragment. The fragment containing CCR5-FLAG-CTC was first digested with NheI and then ligated into pBluebac4.5, which had been blunted with Klenow. This final construct is identified as CCR5-FLAG. This construct was confirmed by restriction digestion and sequenced using standard techniques. This construct was used for expression and was determined to express enough and active form for use in affinity purification screening.
[0060]
(Construction of CCR5 having C-terminal GST tag)
PCR was performed using CCR5 / pcDNA3 (Receptor Biology) as template and primers GST-BamHI and GST-NdeI. The first primer mutated the stop codon of CCR5 to a BamHI restriction site (in-frame using the FLAG / GST tag of pESP-3). A second primer was introduced at the unique NdeI site at the start codon of CCR5. The PCR product was digested with BamHI and NdeI and ligated into similarly digested pESP-3 (a yeast expression vector). This construct is called pCP8. PCP8 was then digested with NdeI and SmaI and filled with Klenow fragment. The fragment containing CCR5-GST was ligated into pBluebac4.5, which was first digested with BglII and then blunted with Klenow. This final construct is identified as pCP10. When describing this protein, this construct is identified as CCR5-GST. This construct was confirmed by restriction digestion and sequenced by standard techniques. This construct was used for expression and was determined to express enough and active form for use in affinity purification screening.
[0061]
Vectors for the three new constructs (CCR5-FLAG, CCR5-GST and CCR5-HIS) were used to co-transfect Sf9 cells for production of each virus stock. These virus stocks were purified using a standard plaque assay and then used in experiments to infect for optimization of expression of CCR5 with various C-terminal tags. High Five cells (Invitrogen) were also transfected with these CCR5-tagged constructs and tested for CCR5 expression. All constructs were determined to express the appropriate tagged receptor. The expression level after 72 hours was about 5-fold higher in High Five cells than in Sf9 cells. All the above experiments were performed using standard techniques known to those skilled in the art.
[0062]
A fourth construct for expression of CCR5 was made from the starting vector pBlueBac4.5 (Invitrogen) to remove the thrombin and enterokinase cleavage sites in the previously described vectors. The GST tag was added to the multiple cloning site by using PCR to purify the GST tag and then ligated into the digested vector (SmaI / EcoRI) using standard methods known to those skilled in the art. This vector was then made compatible with the Gateway technology from Lifetech for ease of manipulation. This was done by ligating SmaI to a cassette (from Lifetech) containing the necessary recombination sites for this technique. CCR5 was amplified using PCR with primers to extend the gene containing the binding site for recombination. The PCR product was then incorporated into a baculovirus vector using BP clonase (enzyme required for homologous recombination) to contain a baculo containing CCR5 with no C-terminal GST tag without an enterokinase or thrombin cleavage site. A vector for virus expression was made. This vector was co-transfected into Sf9 cells for the preparation of the necessary viral stock for expression. The virus was plaque purified and PCR and sequence checked clones were used for CCR5 expression. The time course of this construct indicated that less proteolysis of the protein was observed and that less time was required to obtain maximum expression of the receptor.
[0063]
(B. Activity of CCR5)
Each tagged CCR5 gene (CCR5-GST, CCR5-FLAG and CCR5-HIS) was expressed in Sf9 cells and High Five cells as described herein. All cells from all Sf9 and High Five cell lines were lysed using a hypotonic buffer (10 mM Tris (pH 7.4), 5 mM EDTA), and the membrane preparations were washed. Prepared by homogenization and centrifugation using standard techniques known to the supplier. The membrane preparations for CCR5-GST, CCR5-FLAG and CCR5-HIS were each assayed using a standard radioligand binding assay. Binding assays were performed with 5 μg of membrane in 50 mM Hepes, pH 7.5, 1% BSA. The radioligand MIP1-β (obtained from New England Nuclear, “NEN”) was obtained at room temperature with cold MIP1α (competitive ligand; the natural ligands for CCR5 are RANTES, MIP1β and MIP1α) and at room temperature without cold MIP1α. Time incubated with the membrane, filtered, washed, and bound radioactivity counts were measured using a scintillation counter. Untransfected cells were used as controls in this experiment. The activity of the membrane preparation is determined by the receptor biology (KIP for MIP1-β binding) with at least 20% active protein. _d <1 nM) was comparable to the membrane preparation obtained.
[0064]
(C. Solubilization of CCR5)
Both lysed whole cell and membrane preparations were used for solubilization. Solubilization of the tagged versions of CCR5 (CCR5-FLAG, CCR5-GST and CCR5-HIS) is accomplished at pH in the range of about 6.8 to about 8.2 with detergents (eg, NP-40, Triton X-). 100, β-D-maltoside, n-octylglucoside, CYMAL, Zwittergents, Tween-20, lysophosphatidylcholine, CHAPS, etc.), salts (eg, NaCl, CaCl ₂ , MgCl ₂ , MnCl ₂ , KCl, etc.) and buffers (eg, Tris, Hepes, Hepps, Pipes, Mes, Mops, acetate, phosphate, imidazole, etc.). Conditions for optimal solubilization have been found using Zwittergents 3-14 and low salts (e.g., low magnesium and calcium, no NaCl (0.0 nM NaCl) at pH 8.1. In at least 20% of the solubilized, immobilized protein is active In a highly preferred embodiment, at least 30%, 40%, 50%, and 75% of the solubilized immobilized protein is active It is.
[0065]
(D. Immobilization of CCR5)
After solubilization, both CCR5-GST and CCR5-FLAG were immobilized on an affinity column for use as an active protein for purification and for screening peptide libraries. A schematic showing the immobilization of a GPCR for affinity purification from a library is shown in FIG. CCR5-GST was bound to glutathione-agarose (Pierce) and glutathione-sepharose (Amersham Pharmacia Biotech) and immobilized, and CCR5-FLAG was converted to a specific antibody column that recognizes the FLAG epitope (M2 column, Sigma). Immobilized. Immobilization of the protein in the active form required a surfactant to maintain activity while on the column, as well as an appropriate pH and appropriate salt conditions. This activity was measured by radioactive binding using competition with radiolabeled MIP-1β (used in the membrane assay described above) and cold MIP-1α. Untransfected cells were used as a control for this activity, as well as the column alone. These experiments demonstrated the ability to immobilize microgram quantities of receptor in purified form (sufficient for affinity purification screening) on the resin in active form.
[0066]
(E. Peptide library synthesis)
The library was synthesized on ABI 433A with a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group (Applied Biosystems, Forster City, CA) using Rink Amide MBHA resin (substitution: 0.54 mmol / gm). When a mixture of amino acids is used to degenerate positions, approximately equal amounts of amino acid linkages are obtained by adjusting the amount of amino acids empirically after taking into account literature values (eg, See Ivanetic et al., Combinatorial Chemistry, Vol. 267, Academic Press, San Diego, CA USA, 247-260 (1996)). The binder was HBTU / HOBT / DIEA (1 equivalent per equivalent peptide). Cleavage was performed with a cocktail (82% TFA, 5% phenol, 5% thioanisole, 2.5% 1,2-ethanedithiol, 5% water). The peptide was precipitated from methyl tertiary butyl ether. The library was characterized by MALDI-TOF MS (Louisiana State University) and amino acid sequencing.
[0067]
The first library used a single, degenerate basic base amino acid (i.e., MXXXXXXWXXXXXXXAKKKK). . Through the use of these first libraries, the optimal residues at some or all degenerate positions were defined. A second library was created if not all positions were defined, thereby fixing the defined positions.
[0068]
(F. Screening of peptide library using immobilized CCR5)
Using proteins immobilized on a particular resin (eg, immobilizing CCR5-GST on glutathione-sepharose or CCR5-FLAG on M2 antibody-agarose), incubating a screen of countless compounds is incubated together. And allowing natural selection and binding affinity to purify the ligands preferred by CCR5. These experiments were performed using the linear library 4P4 (+) and other libraries.
[0069]
(G. Phage display)
As an alternative or additional method useful in screening, immobilized functional CCR5 was used to isolate phage that bind to CCR5 using standard techniques known to those of skill in the art. Background subtraction from the glutathione-Sepharose beads, BSA and MIP1β used in the assay was performed by incubating the phage library with a mixture of these compounds. After incubation of the subtracted phage library (ie, NEB, PhD C7C) with the receptor, bound phage were eluted with both the native CCR5 ligand (MIP-1β) and glycine, pH 2.2. PhD C7C is a specialized phage library with seven random amino acids between disulfides and can be obtained from New England Biolabs ("NEB"). Multiple screenings were performed. Both conditions provided specific sequences that could bind to CCR5 and inhibit ligand binding.
[0070]
(Example 4: Preparation and analysis of tagged CXCR4 and library screening thereof)
(A. Cloning and expression of CXCR4)
CXCR4 was isolated from a bisected spleen cDNA library and spliced together. These two fragments were isolated using PCR technology and primers for the 3 'and 5' ends and the middle of the CXCR4 gene. The full-length clone was not isolated using the 3 'and 5'primers; however, two halves were isolated and ligated together using unique BamHI sites within the gene. The identity of the construct was confirmed by sequencing. An alternative splice truncated form was also isolated. This is called CXCR4 shortened type. Tags were added to the C-terminus of the receptor and used to immobilize them for affinity purification assays using standard techniques. The following is a specific example from an experiment using this tagging method.
[0071]
(Construction of CXCR4 having C-terminal histidine tag (insect selective expression system))
Previous constructs containing the gene for GnRHR (gonadotropin releasing hormone receptor) were used to create the first CXCR4 construct. The gene for GnRHR was spliced and replaced with the isolated cDNA for CXCR4. This vector was originally a pet30a vector with a 6 × His tag at the C-terminus.
[0072]
(Construction of CXCR4 construct having C-terminal FLAG tag)
PCR was performed using primers 5'BspE1 CXCR4 and 3'Bgl CXCR4 and a unique site for ligation of the in-frame CXCR4 with FLAG tag of pFLAG-CTC (bacterial expression vector) from Sigma. Operated. This construct is called CXCR4-FLAG-CTC. CXCR4-FLAG was then removed by digestion and filled with Klenow fragment. The fragment containing CXCR4-FLAG was first digested and then ligated into Klenow blunted pBluebac 4.5. This final construct is called CXCR4-FLAG. This construct was confirmed by restriction digestion and sequenced using standard techniques. This construct was used for expression and was determined to express enough and active form for use in affinity purification screening.
[0073]
(Construction of CXCR4 construct having C-terminal GST tag)
The newly constructed CXCR4-FLAG cDNA was removed from the CTC vector and subcloned into another construct (CCR5-GST) instead of CCR5 (using Bgl and BspE1). This created the vector for CXCR4-GST using one step. Constructs were confirmed by restriction digestion and sequenced using standard techniques. This construct was used for expression and was determined to express enough and active form for use in affinity purification screening.
[0074]
(Construction of CXCR4 having N-terminal 6 × His tag)
This construct was prepared by subcloning CXCR4 into a commercially available vector, pBluebacHis2b (Invitrogen). This construct was confirmed by restriction digestion and sequencing using standard techniques.
[0075]
Vectors of three new constructs, CXCR4-FLAG, CXCR4-GST, and CXCR4-HIS, were used to co-transfect Sf9 cells for production of each virus stock. These virus stocks were purified using a standard plaque assay and then used in infection experiments for optimizing expression of CXCR4 with its various C-terminal tags. High Five cells (Invitrogen) were also transfected with these CXCR4-tagged constructs and tested for CXCR4 expression. All constructs were determined to express the appropriately tagged receptor. The expression level after 72 hours in High Five cells was 5 times higher than in Sf9 cells.
[0076]
(B. Activity of CXCR4)
Each tagged CXCR4 gene (CXCR4-FLAG, CXCR4-GST, and CXCR4-HIS) was used to co-transfect Sf9 and High Five cells as described herein. Whole cells from Sf9 and High Five cell lines are lysed using hypotonic buffer (10 mM Tris (pH 7.4), 5 mM EDTA) and membrane preparations are prepared using standard techniques known to those skilled in the art. Prepared by homogenization and centrifugation using Membrane preparations for CXCR4-GST, CXCR4-FLAG and CXCR4-HIS were assayed using a standard radioligand binding assay. Radioligand [ ¹²⁵ I] -SDR-1 (NEN) was incubated with the membrane for 1 hour at room temperature with and without cold SDF-1 (a competing natural ligand), filtered, washed and bound Radioactivity counts were detected using scintillation counting. Untransfected cells were used as controls in this experiment. The activity of the membrane preparation resulted in at least 20% active protein.
[0077]
(C. Solubilization of CXCR4)
Both lysed whole cells and membrane preparations were used for solubilization. Solubilization of tagged versions (CXCR4-FLAG, CXCR4-GST and CXCR4-HIS) on CXCR4 was accomplished by adding detergents (eg, NP-40, Triton X-100, β-D-maltoside, n-octylglucoside, CYMAL, Zwittergents, Tween-20, lysophosphatidylcholine, CHAPS, etc.), salts (eg, NaCl, CaCl ₂ , MgCl ₂ , MnCl ₂ , KCl, etc.) and buffers (eg, Tris, Hepes, Hepps, Pipes, Mes, Mops, acetate, phosphate, imidazole, etc.) to a pH in the range of about 6.8 to about 8.2. And performed. Conditions for optimal solubilization have been found using Zwittergents 3-14 and low salts (e.g., low magnesium and calcium, no NaCl (0.0 nM NaCl) at pH 8.1. In at least 20% of the solubilized, immobilized protein is active In a highly preferred embodiment, at least 30%, 40%, 50%, and 75% of the solubilized, immobilized protein is: Active.
[0078]
(D. Immobilization of CXCR4)
After solubilization, CXCR4-GST was immobilized on an affinity column for use as an active protein for purification and for use in screening peptide libraries. A schematic diagram showing the immobilization of a GPCR for affinity purification from a library is shown in FIG. CXCR4-GST was conjugated and immobilized on glutathione-agarose (Pierce) and glutathione-sepharose (Amersham Pharmacia Biotech). Immobilization of the active form of the protein required the use of detergents and appropriate pH and salt concentrations to maintain the active form while placed on the column. This activity was determined by radiolabeled SDF-1 (if with the membrane assay described above) radioactivity and competition with cold SDF-1. Untransfected cells were used as a control for this activity, as well as the column alone. These experiments demonstrated the ability to immobilize microgram quantities of receptor in purified form sufficient for affinity purification screening on the resin in active form.
[0079]
(E. Peptide library synthesis)
The library was synthesized on ABI 433A with a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group (Applied Biosystems, Forster City, CA) using Rink Amide MBHA resin (substitution: 0.54 mmol / gm). When a mixture of amino acids is used for degenerate positions, approximately equivalent binding of amino acids was obtained by adjusting the amount of amino acids empirically after taking into account literature values (eg, See Ivanetic et al., Combinatorial Chemistry, Vol. 267, Academic Press, San Diego, CA USA, 247-260 (1996)). The binder was HBTU / HOBT / DIEA (1 equivalent per equivalent peptide). Cleavage was performed with a cocktail (82% TFA, 5% phenol, 5% thioanisole, 2.5% 1,2-ethanedithiol, 5% water). The peptide was precipitated from methyl tertiary butyl ether. The library was characterized by MALDI-TOF MS (Louisiana State University) and amino acid sequencing.
[0080]
The first library uses a single, non-degenerate basic base amino acid (i.e., MXXXXXWXXXXXXXAKKKK). did. Through the use of these first libraries, the optimal residues at some or all degenerate positions were defined. A second library was created if not all positions were defined, thereby fixing the defined positions.
[0081]
F. Screening of Peptide Libraries Using Immobilized CXCR4 Proteins are immobilized on a specific resin (eg, CXCR4-GST is immobilized on Glutathione-Sepharose) and screening for countless compounds is incubated together And allowing natural selection and binding affinity to purify the preferred ligand for CXCR4. These experiments were performed using 10 libraries. These experiments can then be performed with other libraries.
[0082]
(G. Phage display)
As an alternative or additional method useful in screening, immobilized functional CXCR4 was used to isolate phage that bind to CXCR4 using standard techniques known to those of skill in the art. Background subtraction from the glutathione-Sepharose beads, BSA and SDF1α used in the assay was performed by incubating the phage library with a mixture of these compounds. After incubation of the subtracted phage library (ie, NEB PhD C7C) with the receptor, bound phage were eluted with both native CXCR4 ligand (SDF1α) and glycine, pH 2.2. PhD C7C is a specialized phage library with seven random amino acids between disulfides and can be obtained from New England Biolabs ("NEB"). Multiple screenings were performed. Both conditions provided specific sequences that could bind to CXCR4 and inhibit ligand binding.
[0083]
Example 5: Prevention or treatment of diseases and disorders
However, during the production, formulation and use of therapeutic peptides, peptidomimetics, or small agonists or antagonists of GPCR binding used for the prevention and treatment of diseases and disorders (eg, AIDS and HIV infection) Presents several complications. Biologically relevant antagonists or agonists that minimize the cost and technical difficulty of commercial production of GPCR therapeutic binding compounds are further contemplated by the present invention. In addition, biologically relevant antagonists or agonists of GPCR binding that do not confer an immunological response to the antagonist or agonist, thereby preventing its effectiveness, are contemplated by the present invention. In addition, suitable formulations that provide a commercially reasonable expiration date of the produced GPCR binding antagonist or agonist without significant loss of biological potency are contemplated herein. Further, useful doses for administration to an individual, suitable for the prevention and treatment of diseases and conditions such as AIDS and HIV infection, are contemplated in the present invention.
[0084]
The identification of suitable candidates, alone or mixed with other suitable molecules, that are competent for inhibiting GPCR binding is contemplated by the present invention. Such candidates are further intended to produce commercially significant quantities of the above identified candidates that are biologically suitable for the prevention and treatment of diseases and conditions such as AIDS and HIV infection. I do. In addition, the present invention relates to a method for reducing the therapeutic quality of a GPCR binding antagonist, agonist or derivative in which any undesired properties of the initially identified analog (eg, toxicity in vivo or propensity to degrade upon storage) are reduced. Provides commercially significant quantities of production.
[0085]
After identification and design of peptides, peptidomimetics, or small molecule antagonists of GPCR binding activity, methods of preventing and treating diseases such as AIDS and HIV infection inhibit GPCR binding as described herein. And administering a composition comprising the compound. Administration can be by any suitable route. Thus, where appropriate, administration can include oral or parenteral, including intravenous and intraperitoneal routes of administration. A particularly preferred method is by sustained release injection of the appropriate formulation. Further, administration can be by periodic injection of a bolus of the composition or intravenously from a reservoir that is extracorporeal (eg, an intravenous bag) or internal (eg, a bioerodable implant). Or it may be made more continuous by intraperitoneal administration.
[0086]
The therapeutic compositions contemplated by the present invention can be administered by any suitable means, either directly (eg, locally, such as by injection, implantation or local administration to a tissue site) or systemically (eg, parenterally). (Orally or orally) to the individual. The composition can be administered parenterally (e.g., intravenously, subcutaneously, intramolecularly, ocular, intraperitoneally, intramuscularly, buccal, rectum, vaginal, intraorbitally, intracervical, intracranial, intraspinal, chamber, intrathecal, When provided by a cisterna magna, intracapsular, intranasal) or by aerosol administration, the composition may include portions of an aqueous or physiologically compatible liquid suspension or solution. Thus, the carrier or vehicle is physiologically acceptable so that, in addition to delivering the desired composition to the subject, does not have other adverse effects on the patient's electrolyte and / or volume balance.
[0087]
Solutions useful for parenteral administration are prepared by any of the methods well known in the art of pharmacy, for example, those described in REMINGTON'S PHARMACEUTICAL SCIENCE (Gennar, A., eds.), Mack Pub., 1990. obtain. Formulations of the therapeutic agents of the present invention can include, for example, polyalkylene glycols (eg, polyethylene glycol, oils of vegetable origin, hydrogenated naphthalene, and the like). In particular, formulations for direct administration may include glycerol and other high viscosity compositions that help maintain the drug at the desired site. Biocompatible, preferably bioresorbable polymers (eg, including hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide and glycolide polymers and lactide / glycolide copolymers) control drug release in vivo Can be a useful excipient. The concept of sustained-release injectable formulations for peptide drugs is well-accepted and offers several advantages. First, for example, bioavailability is high. Second, the treatment regimen may consist of once a month or three months (like Leupron® from Abott) or once a year (eg, Viadur® from Alza). Third, sustained-release injectable formulations substantially reduce the dose that can be used (the Leupron injection dose is 1 mg / day, but the 90-day formulation usage is less than the total 11.25 mg). Also, increased efficacy can be achieved when treatments are sequentially present to prevent infectivity. This idea is particularly important in view of the need to achieve cure of the disease by preventing the remodeling of the slow-to-clear deposits of the infection (eg, the memory T cell compartment). For example, Lee, V .; Ed., Peptide and Protein Drug Delivery, Marcel Dekker, Inc. , NY (1991).
[0088]
Other potentially useful parenteral delivery systems for these agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhaled administration include, for example, lactose as an excipient, or an aqueous solution (including, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate) or nasal drops or intranasal It can be an oily solution for administration in the form of a gel to be applied to a liquid. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for colonic administration, or citric acid for vaginal administration.
[0089]
Further aspects and embodiments of the present invention will be apparent to those skilled in the art.
[Brief description of the drawings]
FIG. 1 shows a peptide library with immobilized non-denatured lysine or arginine, as well as eight denatured positions consisting of approximately equal proportions of 18 amino acids.
FIG. 2 shows screening of a peptide library using a binding domain.
FIG. 3 shows the immobilization of GPCRs for affinity purification from a library.

Claims (32)

A method for identifying a compound that binds to a G protein-coupled receptor, comprising:
a) providing a library of two or more molecules;
b) providing a molecule having binding properties corresponding to a G protein-coupled receptor, wherein the molecule is bound to a support;
c) binding a molecule from the library of two or more molecules to the molecule having binding properties corresponding to a G protein-coupled receptor bound to the support;
d) separating the bound molecule from the molecule bound to the support; and e) identifying the bound molecule as a binding compound for a G protein-coupled receptor.
A method comprising: The method of claim 1, wherein the library of two or more molecules is selected from the group consisting of linear peptides, cyclic peptides, natural amino acids, unnatural amino acids, peptidomimetic compounds, and low molecular weight compounds. 2. The method of claim 1, wherein said molecule having binding properties corresponding to a G protein-coupled receptor is a partially purified G protein-coupled receptor. 2. The method of claim 1, wherein at least one of the two or more molecules is selected from the group consisting of a peptide, peptidomimetic, or small molecule capable of replacing a protein capable of binding to a receptor, enzyme, or other protein. the method of. 2. The method of claim 1, further comprising the step of dissolving the molecule having binding properties corresponding to a G protein-coupled receptor in the substantial absence of sodium chloride. 2. The method of claim 1, further comprising using a buffer having a low salt concentration to dissolve the molecule having binding properties corresponding to a G protein-coupled receptor. 2. The method of claim 1, wherein at least one of said two or more molecules comprises a molecule having an antagonistic effect on G protein-coupled receptor binding activity. 2. The method of claim 1, wherein said library comprises a phage library. The method of claim 1, wherein steps a, b, c, and d are repeated at least once before step e. The method of claim 1, wherein the molecule having binding properties corresponding to a G protein-coupled receptor comprises a G protein-coupled receptor molecule and a tag selected from the group consisting of: GST, FLAG, 6 × His, C -MYC, MBP, V5, Xpress, CBP, and HA. A binding compound for a G protein-coupled receptor identified according to the method of claim 1. A method of preventing a disease or disorder in a patient, the method comprising administering to the patient a therapeutic composition comprising a compound of claim 11 in a physiological carrier. 13. The method of claim 12, wherein the disease or disorder is an HIV infection. 13. The method according to claim 12, wherein said disease or disorder is AIDS. A method of treating or preventing a disease or disorder in a patient, comprising administering to the patient a therapeutic composition comprising a compound of claim 11 in a sustained release injectable formulation. Including, methods. 16. The method of claim 15, wherein said disease or disorder is an HIV infection. 16. The method of claim 15, wherein said disease or disorder is AIDS. A computer assisted method for identifying the relative binding affinity of a test molecule for a G protein-coupled receptor, comprising:
a) inputting input data characterizing a G protein-coupled receptor into a computer program;
b) inputting input data characterizing at least one test peptide-like molecule whose respective sequences are known, but whose binding affinity is unknown;
c) analyzing each test peptide-like molecule applied using the computer program to generate a prediction of relative binding affinity for each test peptide-like molecule, and outputting such a prediction;
A method comprising: A method for determining an amino acid sequence motif of an interaction site of a binding compound for a G protein-coupled receptor, comprising:
a) A molecule and a peptide library having a binding property corresponding to a G protein-coupled receptor are subjected to a condition allowing interaction between the molecule having a binding property corresponding to the G protein-coupled receptor and the peptide library. And contacting;
b) A molecule having a binding property corresponding to the G protein-coupled receptor interacts with the peptide library, and as a result, a molecule having a binding property corresponding to the G protein-coupled receptor and a molecule corresponding to the G protein-coupled receptor Forming a complex between a molecule having binding properties and a subpopulation of library members that can interact with the molecule;
c) a member of a library that cannot interact with a molecule having a binding property corresponding to the G protein-coupled receptor from a member of a library that can interact with a molecule having a binding property corresponding to the G protein-coupled receptor Isolating the subpopulation;
d) linearizing said subpopulation of library members capable of interacting with molecules having binding properties corresponding to said G protein-coupled receptor;
e) determining the relative abundance of a different amino acid residue at each degenerate position in the mixture of members of the linearized library; and f) determining the linearity of the linearized library. Determining an amino acid sequence motif of an interaction site of a molecule having a binding property corresponding to the G protein-coupled receptor based on the relative abundance of a different amino acid residue at each degenerate position in the mixture of members. ,
A method comprising: An amino acid sequence motif of a binding compound for a G protein-coupled receptor, identified according to the method of claim 19. 20. A binding compound having an amino acid sequence motif for a G protein-coupled receptor, determined by the method of claim 19. 20. The method of claim 19, wherein at least one member of said peptide library comprises at least one unnatural amino acid. 20. The molecule according to claim 19, wherein the molecule having a binding property corresponding to the G protein-coupled receptor is selected from the group consisting of a linear peptide, a cyclic peptide, a natural amino acid, an unnatural amino acid, a peptidomimetic compound, and a small molecule compound. the method of. 20. The method of claim 19, wherein said peptide library comprises at least one molecule selected from the group consisting of linear peptides, cyclic peptides, natural amino acids, unnatural amino acids, peptidomimetic compounds, and low molecular weight compounds. A library comprising members based on an amino acid sequence motif of an interaction site of a binding compound for a G protein-coupled receptor, wherein the motif is a compound wherein at least one peptide member from a peptide library binds to the G protein-coupled receptor. A library that is capable of interacting with a G protein-coupled receptor and is determined by determining the amino acid sequence of at least one peptide that interacts with a binding compound for the G protein-coupled receptor. A method for solubilizing and immobilizing a compound corresponding to the binding property of a G protein-coupled receptor, wherein when the compound corresponding to the binding of the G protein-coupled receptor is determined, the solubilization and the immobilization are substantially performed. A method performed in the absence of sodium chloride. A method for solubilizing and immobilizing a compound corresponding to the binding property of a G protein-coupled receptor, wherein when the compound corresponding to the binding of the G protein-coupled receptor is determined, the solubilization and immobilization may be performed at a low salt concentration. The method is performed by using. 28. The method of claim 27, wherein said low salt concentration comprises a predetermined amount of magnesium and calcium. A constructed G protein-coupled receptor transfer vector, wherein the transfer vector comprises a G protein-coupled receptor molecule and a tag selected from the group consisting of: GST, FLAG, 6 × His, C-MYC, MBP , V5, Xpress, CBP, and HA. A method of using a three-dimensional structure of a G protein-coupled receptor in a drug screening assay, comprising:
a) selecting a potential drug by performing a rational drug design using the three-dimensional structure, wherein the selecting step is performed in combination with computer modeling;
b) contacting the potential drug with a first molecule comprising a first G protein-coupled receptor; and c) detecting binding of the potential drug to the first molecule;
Wherein the potential drug is selected as the drug if the potential drug binds to the first molecule. 31. The method of claim 30, wherein said first molecule is labeled. 31. The method of claim 30, wherein said first molecule is attached to a solid support.

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